Oncolytic rhabdovirus

ABSTRACT

Embodiments of the invention include compositions and methods related to non-VSV rhabdoviruses and their use as anti-cancer therapeutics. Such rhabdoviruses possess tumor cell killing properties in vitro and in vivo.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/884,967, filed May 27, 2020, which is a continuation of U.S.application Ser. No. 16/101,265, filed Aug. 10, 2018, now abandoned,which is a continuation of U.S. application Ser. No. 15/436,520, filedFeb. 17, 2017, now abandoned, which is a continuation of U.S.application Ser. No. 13/937,043, filed Jul. 8, 2013, now U.S. Pat. No.9,572,883, which is a continuation of U.S. application Ser. No.12/441,494 filed Oct. 21, 2010, now U.S. Pat. No. 8,481,023, which is aU.S. national stage of International Patent Application No.PCT/IB2007/004701, filed Sep. 17, 2007, which claims the benefit of U.S.Provisional Application No. 60/844,726 filed Sep. 15, 2006, each ofwhich is hereby incorporated by reference herein in its entirety.

SEQUENCE LISTING

This application incorporate by reference in its entirety the ComputerReadable Form (“CRF”) of a Sequence Listing in ASCII text formatsubmitted via EFS-Web. The Sequence Listing text file submitted viaEFS-Web is entitled “14596-063-999_Substitute_Seqlisting.txt,” wascreated on Feb. 9, 2021 and is 174,245 bytes in size.

I. FIELD OF THE INVENTION

This invention relates generally to virology and medicine. In certainaspects the invention relates to oncolytic viruses, particularly non-VSVoncolytic rhabdoviruses and oncolytic rhabdoviruses comprising a non-VSVglycoprotein.

II. BACKGROUND

A number of viruses have been shown to replicate in and kill a widevariety of tumor cells in vitro (Sindbis virus (Unno et al., 2005);Sendai virus (Kinoh et al., 2004); Coxackie virus (Shafren et al.,2004); Herpes simplex virus (Mineta et al., 1995); Parvovirus (Abschuetzet al., 2006); Adenovirus (Heise et al., 2000); Polio virus (Gromeier etal., 2000); Newcastle disease virus (Sinkovics and Horvath, 2000);Vesicular stomatitis virus (Stojdl et al., 2000); Measles virus (Groteet al., 2001); Reovirus (Coffey et al., 1998); Retrovirus (Logg et al.,2001); Vaccinia (Timiryasova et al., 1999); and Influenza (Bergmann etal., 2001)). In addition, such viruses have demonstrated efficacy intreating animal models of cancer.

Vesicular stomatitis virus (VSV), a well known and well studiedrhabdovirus, has been shown to kill tumor cell lines in cell cultureexperiments, and has demonstrated efficacy in a variety of rodent cancermodels (Stojdl et al., 2000; Stojdl et al., 2003). However, VSV does notkill all cancer cells.

SUMMARY OF THE INVENTION

Several newly identified rhabdoviruses are much more efficient atkilling particular cancers or cancer cell lines than VSV. Also, VSV andattenuated mutants of VSV are neurovirulent and cause CNS pathology inrodents and primates. Several rhabdoviruses do not infect the CNS (i.e.,Muir Springs and Bahia Grande: Kerschner et al., 1986), and demonstratea more acceptable safety profile. In addition, therapies based on thenovel rhabdoviruses can be used to treat cancers of the CNS, bothprimary and secondary. The rhabdoviruses of the invention (and/or otheroncolytic agents) can be used in succession to bypass the host immuneresponse against a particular therapeutic virus(es). This would allowprolonged therapy and improve efficacy.

Embodiments of the invention include compositions and methods related tonon-VSV rhabdoviruses and their use as anti-cancer therapeutics. Suchrhabdoviruses possess tumor cell killing properties in vitro and invivo.

As used herein, a non-VSV rhabdovirus will include one or more of thefollowing viruses or variants thereof: Arajas virus, Chandipura virus,Cocal virus, Isfahan virus, Maraba virus, Piry virus, Vesicularstomatitis Alagoas virus, BeAn 157575 virus, Boteke virus, Calchaquivirus, Eel virus American, Gray Lodge virus, Jurona virus, Klamathvirus, Kwatta virus, La Joya virus, Malpais Spring virus, Mount Elgonbat virus, Perinet virus, Tupaia virus, Farmington, Bahia Grande virus,Muir Springs virus, Reed Ranch virus, Hart Park virus, Flanders virus,Kamese virus, Mosqueiro virus, Mossuril virus, Barur virus, Fukuokavirus, Kem Canyon virus, Nkolbisson virus, Le Dantec virus, Keuralibavirus, Connecticut virus, New Minto virus, Sawgrass virus, Chaco virus,Sena Madureira virus, Timbo virus, Almpiwar virus, Aruac virus, Bangoranvirus, Bimbo virus, Bivens Arm virus, Blue crab virus, Charlevillevirus, Coastal Plains virus, DakArK 7292 virus, Entamoeba virus, Garbavirus, Gossas virus, Humpty Doo virus, Joinjakaka virus, Kannamangalamvirus, Kolongo virus, Koolpinyah virus, Kotonkon virus, Landjia virus,Manitoba virus, Marco virus, Nasoule virus, Navarro virus, Ngainganvirus, Oak-Vale virus, Obodhiang virus, Oita virus, Ouango virus, ParryCreek virus, Rio Grande cichlid virus, Sandjimba virus, Sigma virus,Sripur virus, Sweetwater Branch virus, Tibrogargan virus, Xiburemavirus, Yata virus, Rhode Island, Adelaide River virus, Berrimah virus,Kimberley virus, or Bovine ephemeral fever virus. In certain aspects,non-VSV rhabdovirus can refer to the supergroup of Dimarhabdovirus(defined as rhabdovirus capable of infecting both insect and mammaliancells). In specific embodiments, the rhabdovirus is not VSV. Inparticular aspects the non-VSV rhabdovirus is a Carajas virus, Marabavirus, Farmington, Muir Springs virus, and/or Bahia grande virus,including variants thereof.

One embodiment of the invention includes methods and compositionscomprising an oncolytic non-VSV rhabdovirus or a recombinant oncolyticnon-VSV rhabdovirus encoding one or more of rhabdoviral N, P, M, Gand/or L protein, or variant thereof (including chimeras and fusionproteins thereof), having an amino acid identity of at least or at most20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, 98, 99, 100%,including all ranges and percentages there between, to the N, P, M, Gand/or L protein of Arajas virus, Chandipura virus, Cocal virus, Isfahanvirus, Maraba virus, Piry virus, Vesicular stomatitis Alagoas virus,BeAn 157575 virus, Boteke virus, Calchaqui virus, Eel virus American,Gray Lodge virus, Jurona virus, Klamath virus, Kwatta virus, La Joyavirus, Malpais Spring virus, Mount Elgon bat virus, Perinet virus,Tupaia virus, Farmington, Bahia Grande virus, Muir Springs virus, ReedRanch virus, Hart Park virus, Flanders virus, Kamese virus, Mosqueirovirus, Mossuril virus, Barur virus, Fukuoka virus, Kern Canyon virus,Nkolbisson virus, Le Dantec virus, Keuraliba virus, Connecticut virus,New Minto virus, Sawgrass virus, Chaco virus, Sena Madureira virus,Timbo virus, Almpiwar virus, Aruac virus, Bangoran virus, Bimbo virus,Bivens Arm virus, Blue crab virus, Charleville virus, Coastal Plainsvirus, DakArK 7292 virus, Entamoeba virus, Garba virus, Gossas virus,Humpty Doo virus, Joinjakaka virus, Kannamangalam virus, Kolongo virus,Koolpinyah virus, Kotonkon virus, Landjia virus, Manitoba virus, Marcovirus, Nasoule virus, Navarro virus, Ngaingan virus, Oak-Vale virus,Obodhiang virus, Oita virus, Ouango virus, Parry Creek virus, Rio Grandecichlid virus, Sandjimba virus, Sigma virus, Sripur virus, SweetwaterBranch virus, Tibrogargan virus, Xiburema virus, Yata virus, RhodeIsland, Adelaide River virus, Berrimah virus, Kimberley virus, or Bovineephemeral fever virus. Any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 12 13, 14,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or more,including all integers or ranges there between, of these virus can bespecifically excluded from the claim scope. VSV or any non-VSVrhabdovirus can be the background sequence into which a variantG-protein or other viral protein can be integrated.

In another aspect of the invention, a non-VSV rhabdovirus, or arecombinant there of, can comprise a nucleic acid segment encoding atleast or at most 10, 20, 30, 40, 45, 50, 60, 65, 70, 80, 90, 100, 125,175, 250 or more contiguous amino acids, including all value and rangesthere between, of N, P, M, G or L protein of one or more non-VSVrhabdovirus, including chimeras and fusion proteins thereof. In certainembodiments a chimeric G protein will include a cytoplasmic,transmembrane, or both cytoplasmic and transmembrane portions of a VSVor non-VSV G protein.

Methods and compositions of the invention can include a secondtherapeutic virus, such as an oncolytic or replication defective virus.Oncolytic typically refers to an agent that is capable of killing,lysing, or halting the growth of a cancer cell. In terms of an oncolyticvirus the term refers to a virus that can replicate to some degree in acancer cell, cause the death, lysis, or cessation of cancer cell growthand typically have minimal toxic effects on non-cancer cells. A secondvirus includes, but is not limited to an adenovirus, a vaccinia virus, aNewcastle disease virus, an alphavirus, a parvovirus, a herpes virus, arhabdovirus, a non-VSV rhabdovirus and the like. In other aspects, thecomposition is a pharmaceutically acceptable composition. Thecomposition may also include a second anti-cancer agent, such as achemotherapeutic, radiotherapeutic, or immunotherapeutic.

Further embodiments of the invention include methods of killing ahyperproliferative cell comprising contacting the cell with an isolatedoncolytic rhabdovirus composition; or

Still further methods include the treatment of a cancer patientcomprising administering an effective amount of an oncolytic rhabdoviruscomposition.

In certain aspects of the invention, a cell may be comprised in apatient and may be a hyperproliferative, neoplastic, pre-cancerous,cancerous, metastatic, or metastasized cell. A non-VSV rhabdovirus canbe administered to a patient having a cell susceptible to killing by atleast one non-VSV rhabdovirus or a therapeutic regime or compositionincluding a non-VSV rhabdovirus. Administration of therapeuticcompositions may be done 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more timeswith 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-VSV rhabdovirus orrecombinant non-VSV rhabdovirus, alone or in various combinations. Thecomposition administered can have 10, 100, 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸,10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or more viral particles or plaqueforming units (pfu). Administration can be by intraperitoneal,intravenous, intra-arterial, intramuscular, intradermal, subcutaneous,or intranasal administration. In certain aspects, the compositions areadministered systemically, particularly by intravascular administration,which includes injection, perfusion and the like. The methods ofinvention can further comprise administering a second anti-cancertherapy, such as a second therapeutic virus. In particular aspects atherapeutic virus can be an oncolytic virus, more particularly a non-VSVrhabdovirus. In other aspects, a second anti-cancer agent is achemotherapeutic, a radiotherapeutic, an immunotherapeutic, surgery orthe like.

Embodiments of the invention include compositions and methods related toa VSV rhabdoviruses comprising a heterologous G protein and their use asanti-cancer therapeutics. Such rhabdoviruses possess tumor cell killingproperties in vitro and in vivo.

As used herein, a heterologous G protein includes non-VSV rhabdovirus.Non-VSV rhabdoviruses will include one or more of the following virusesor variants thereof: Arajas virus, Chandipura virus, Cocal virus,Isfahan virus, Maraba virus, Piry virus, Vesicular stomatitis Alagoasvirus, BeAn 157575 virus, Boteke virus, Calchaqui virus, Eel virusAmerican, Gray Lodge virus, Jurona virus, Klamath virus, Kwatta virus,La Joya virus, Malpais Spring virus, Mount Elgon bat virus, Perinetvirus, Tupaia virus, Farmington, Bahia Grande virus, Muir Springs virus,Reed Ranch virus, Hart Park virus, Flanders virus, Kamese virus,Mosqueiro virus, Mossuril virus, Barur virus, Fukuoka virus, Kern Canyonvirus, Nkolbisson virus, Le Dantec virus, Keuraliba virus, Connecticutvirus, New Minto virus, Sawgrass virus, Chaco virus, Sena Madureiravirus, Timbo virus, Almpiwar virus, Aruac virus, Bangoran virus, Bimbovirus, Bivens Arm virus, Blue crab virus, Charleville virus, CoastalPlains virus, DakArK 7292 virus, Entamoeba virus, Garba virus, Gossasvirus, Humpty Doo virus, Joinjakaka virus, Kannamangalam virus, Kolongovirus, Koolpinyah virus, Kotonkon virus, Landjia virus, Manitoba virus,Marco virus, Nasoule virus, Navarro virus, Ngaingan virus, Oak-Valevirus, Obodhiang virus, Oita virus, Ouango virus, Parry Creek virus, RioGrande cichlid virus, Sandjimba virus, Sigma virus, Sripur virus,Sweetwater Branch virus, Tibrogargan virus, Xiburema virus, Yata virus,Rhode Island, Adelaide River virus, Berrimah virus, Kimberley virus, orBovine ephemeral fever virus. In certain aspects, non-VSV rhabdoviruscan refer to the supergroup of Dimarhabdovirus (defined as rhabdoviruscapable of infection both insect and mammalian cells). In particularaspects the non-VSV rhabdovirus is a Carajas virus, Maraba virus, MuirSprings virus, and/or Bahia grande virus, including variants thereof.

One embodiment of the invention includes methods and compositionscomprising a oncolytic VSV rhabdovirus comprising a heterologous Gprotein or a recombinant oncolytic VSV rhabdovirus encoding one or moreof non-VSV rhabdoviral N, P, M, G and/or L protein, or variant thereof(including chimeras and fusion proteins thereof), having an amino acididentity of at least or at most 20, 30, 40, 50, 60, 65, 70, 75, 80, 85,90, 92, 94, 96, 98, 99, 100%, including all ranges and percentages therebetween, to the N, P, M, G, and/or L protein of a non-VSV rhabdovirus.

In another aspect of the invention, a VSV rhabdovirus comprising aheterologous G protein or recombinant thereof, can comprise a nucleicacid comprising a nucleic acid segment encoding at least or at most 10,20, 30, 40, 45, 50, 60, 65, 70, 80, 90, 100, 125, 175, 250 or morecontiguous amino acids, including all value and ranges there between, ofN, P, M, G, or L protein of a non-VSV rhabdovirus, including chimerasand fusion proteins thereof. In certain aspects, a chimeric G proteinmay comprise a cytoplasmic, transmembrane, or both a cytoplasmic andtransmembrane portion of VSV or a second non-VSV virus or non-VSVrhabdovirus.

Methods and compositions of the invention can include a secondtherapeutic virus, such as an oncolytic or replication defective virus.A second virus includes, but is not limited to an adenovirus, a vacciniavirus, a Newcastle disease virus, a herpes virus, a rhabdovirus, anon-VSV rhabdovirus and the like. In other aspects, the composition is apharmaceutically acceptable composition. The composition may alsoinclude a second anti-cancer agent, such as a chemotherapeutic,radiotherapeutic, or immunotherapeutic.

Further embodiments of the invention include methods of killing ahyperproliferative cell comprising contacting the cell with an isolatedoncolytic rhabdovirus, VSV comprising a heterologous G protein molecule,or a non-VSV rhabdovirus composition. Still further methods include thetreatment of a cancer patient comprising administering an effectiveamount of such a viral composition.

In certain aspects of the invention, a cell may be comprised in apatient and may be a hyperproliferative, neoplastic, pre-cancerous,cancerous, metastatic, or metastasized cell. A virus of the inventioncan be administered to a patient having a cell susceptible to killing byat least one virus or a therapeutic regime or composition including avirus. Administration of therapeutic compositions may be done 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more times with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore virus, alone or in various combinations. The compositionadministered can have 10, 100, 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰,10¹¹, 10¹², 10¹³, 10¹⁴, or more viral particles or plaque forming units(pfu). Administration can be by intraperitoneal, intravenous,intra-arterial, intramuscular, intradermal, subcutaneous, or intranasaladministration. In certain aspects, the compositions are administeredsystemically, particularly by intravascular administration, whichincludes injection, perfusion and the like. The methods of invention canfurther comprise administering a second anti-cancer therapy, such as asecond therapeutic virus. In particular aspects a therapeutic virus canbe an oncolytic virus such as a VSV comprising a heterologous G protein,more particularly a non-VSV rhabdovirus. In other aspects, a secondanti-cancer agent is a chemotherapeutic, a radiotherapeutic, animmunotherapeutic, surgery or the like.

Other embodiments of the invention are discussed throughout thisapplication. Any embodiment discussed with respect to one aspect of theinvention applies to other aspects of the invention as well, and viceversa. The embodiments in the Detailed Description and Example sectionsare understood to be non-limiting embodiments of the invention that areapplicable to all aspects of the invention.

The terms “inhibiting,” “reducing,” or “preventing,” or any variation ofthese terms, when used in the claims and/or the specification includesany measurable decrease or complete inhibition to achieve a desiredresult. Desired results include but are not limited to palliation,reduction, slowing, or eradication of a cancerous or hyperproliferativecondition, as well as an improved quality or extension of life.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Phylogenetic relationships between rhabdoviruses based on a GDEalignment of a relatively conserved region of the N protein (119 aminoacids), and using the paramyxovirus Human parainfluenza virus 1 (HPIV-1)as the outgroup. The tree was generated by the neighbor-joining methodand bootstrap values (indicated for each branch node) were estimatedusing 1000 tree replicas. Branch lengths are proportional to geneticdistances. The scale bar corresponds to substitutions per amino acidsite Courtesy of H. Badrane and P. J. Walker).

FIG. 2. Summary of in vitro tumor cell killing assay. Cells from the NCI60 cell panel were infected for 96 h with a series of dilution ofvarious viruses. Cell viability was assayed using crystal violetstaining to detect residual viable cells. The EC₅₀ was calculated fromthe resulting cell killing curves and summarized in table format. Forclarity, the EC₅₀ values have been converted to a value from 1-7 asdescribed in the legend. In addition, the shading has been used toindicate the EC₅₀ range (i.e., darkest to lightest represents highestEC₅₀ to lowest EC₅₀ values). Viruses are abbreviated as follows: MS=MuirSprings, BG=Bahia Grande, NGG=Ngaingan, TIB=Tibrogargan, FMT=Farmington,MRB=Maraba, CRJ=Carajas, VSVHR=Vesicular Stomatitis Virus HR strain andVV=Vaccinia virus JX-963. This data demonstrates that not allrhabdoviruses are equally oncolytic, in fact closely relatedrhabdoviruses behave very differently on the same tumor cell lines. Thusthere is currently no method to predict which rhabdoviruses haveoncolytic potential. Empirical testing is required to identify goodoncolytic candidate viruses.

FIGS. 3A-3B. Rhabdovirus productivity on tumor cell lines. SNB19 humanglioblastoma and NCI H226 human lung carcinoma cell lines were infectedwith various rhabdoviruses (MOI=3) and monitored over time for virusproduction by plaque assay. The data shows that not all rhabdoviruseshave the same ability to replicate in these tumor cell lines. NCIH226cell reveal a great disparity in virus productivity with Bahia Grandenot producing virus at all while Maraba virus is able to produce copiousinfectious virions.

FIG. 4. Schematic of rescue system to recover recombinant rhabdovirusesfrom plasmid DNA form. In this example, the Maraba virus has been clonedinto a DNA plasmid between the T7 promoter and a rybozyme sequence fromHepatitis D virus. A549 cells are infected with T7 expressing vacciniavirus and then subsequently transfected with a Maraba genome vectorengineered to express GFP. The rescued virions are purified and thenused to infect Vero cells for 24 hours, resulting in GFP expression inthese cells when visualized by fluorescence microscopy.

FIG. 5. Bioselecting improved strains of oncolytic rhabdoviruses.Rhabdoviruses are quasi-species. Bahia Grande is not neuropathogenic buthas the ability to kill human glioblastoma cells. The inventorscontemplated improving its virulence while maintaining its selectivityfor cancer cells. To improve the virulence of a rhabdovirus for a tumorcell, the inventors selected virus mutants with increased replicationcapacity in a human glioblastoma cell line. Briefly, 5×10⁵ SNB19 cellswere infected with 2.5×10⁶ viral particles, giving an MOI of 5. Theinitial inoculum had a volume of 200 μl and was allowed 1 hour to infectbefore the cells were washed 10 times with PBS. The last wash wasanalyzed for viral particles by plaque assay to ensure proper removal ofinput virus. At increasing time points, the entire supernatant wascollected and replaced with fresh media. The collected media was used toinfect new cells for amplification and was analyzed by plaque assay forthe presence of viral particles. For the first passage, collectionsoccurred at 4, 8, 12 and 24 hpi (hours post infection) until the initialtime for viral release was determined. Viruses from the earliest timepoint were amplified back to a population of 10⁶ and then re-passed.

FIG. 6. Bioselecting improved strains of oncolytic rhabdoviruses. Inthis example, Bahia Grande virus underwent up to 6 iterative cycles ofbioselection. The parental strain (WT) along with passages 4-6 weremonitored for virus production in SNB19 cells at 4, 6 and 8 hours postinfection. A clear and progressive improvement in speed of initial virusreplication is evident during increasing rounds of bioselection.MRB=Maraba is included as, an exemplar of rapid and desirable virusreplication in the cancer cell line.

FIG. 7. Bahia Grande P13 underwent 13 rounds of bioselection. This virusdemonstrated improved virus replication not only in the humanglioblastoma used during the bioselection protocol, but on an unrelatedhuman glioblastoma and a human ovarian carcinoma cell line. Thisdemonstrates that rhabdoviruses can be bioselected to improve theironcolytic properties and these improvements are effective on otherdisparate cancers.

FIG. 8. Balb/C mice were infected intracranially with the indicatedviruses and monitored for morbidity and/or mortality. Both wild type VSV(HR strain) and the delta M51 mutant strain of VSV were extremelyneurotoxic, demonstrating hind limb paralysis within days of infection,while Bahia Grande and Muir Springs viruses showed no neurotoxicity.Bahia Grande P6 is a bioselected strain of Bahia Grande with improvedreplication in human glioblastoma cells. This strain also showed noneurotoxicity, demonstrating that rhabdoviruses can be bioselected forimproved virulence on tumor cells, while maintaining their safetyprofile in normal healthy tissue.

FIG. 9. In vivo efficacy of Maraba and Carajas rhabdoviruses compared toChandripura and WT VSV and delta 51 VSV 4T1 tumors (firefly luciferaseexpressing) were established in 5-8 week old Balb/C female mice byinjecting 10⁶ tumor cells in the left, rear mammary gland. After oneweek, mice were injected intravenously on day 1 & 2 (each dose=10⁷ pfuWT VSV, Δ51 GFP VSV, Maraba or Chandipura; or 10⁸ pfu Carajas). Tumorresponses were measured by bioluminescence imaging using an IVIS 200(Xenogen) (measured as photons/s/cm²).

FIG. 10. Infectivity of G-less VSV pseudotyped with Isfahan G and VSV Gprotein.

FIG. 11. A one step growth curve of VSV WT, Isfahan and RVR IsfG1viruses.

FIG. 12. RVR comprising an Isfahan G protein remains oncolytic. Thecytotoxicity of Isfahan virus, VSV d51 and RVR IsfG1 were assessed onvarious cancer cell lines.

FIGS. 13A-13C. RVR comprising Isf G1 is a able to escape immune responseto VSV in vivo. In vivo luciferase detection was used to determine theamount of virus in mice inoculated with RVR IsfG1 or VSV. FIG. 13A, invivo detection of recombinant virus injected into naïve mice. FIG. 13B,in vivo detection of VSV injected into mice immunized with VSV. FIG.13C, in vivo detection of recombinant RVR IsfG1 virus injected into miceimmunized with VSV.

FIG. 14. Virus yields from infected tumors. Tumors were infected withrecombinant virus or VSV in the presence or absence of immunization withVSV (as indicated). Graphed data shows the amount virus resulting fromthe infection of the tumor.

FIG. 15. A one step growth curve of VSV WT, chandipura virus andRVR_(Cha)G¹. Results show that the recombinant produces the same amountof virus as VSV.

FIG. 16. Cytotoxicity of VSV WT, chandipura virus and RVR_(Cha)G¹.Results show that the recombinant is as cytotoxic as VSV.

FIG. 17. A one step growth curve of VSV WT, Maraba virus andRVR_(Mar)G¹. Results show that recombinant virus titer was greater thanVSV at 48 and 72 h.

FIG. 18. Cytotoxicity of VSV WT, Maraba virus and RVR_(Mar)G¹. Resultsshow that both maraba and the RVR_(Mar)G¹ are cytotoxic in tumor cellslines and that they are generally more cytotoxic to tumor cells that VSVWT.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the invention are based on the killing by non-VSV rhabdovirusor pseudotyped rhabdovirus of several kinds or types cancer cells, whichare resistant to killing by VSV. Some of the advantages of theseoncolytic rhabdoviruses and recombinant rhabdoviruses include thefollowing: (1) Antibodies to the inventive rhabdoviruses will be rare tonon-existent in most populations of the world. (2) rhabdovirusesreplicate more quickly than other oncolytic viruses such as adenovirus,reovirus, measles, parvovirus, retrovirus, and HSV. (3) Rhabdovirus growto high titers and are filterable through 0.2 micron filter. (4) Theoncolytic rhabdoviruses and recombinants thereof have a broad hostrange, capable of infecting many different types of cancer cells and arenot limited by receptors on a particular cell (e.g., coxsackie, measles,adenovirus). (5) The rhabdovirus of the invention are amenable togenetic manipulation. (6) The rhabdovirus also has a cytoplasmic lifecycle and do not integrate in the genetic material a host cell, whichimparts a more favorable safety profile.

Embodiments of the invention include compositions and methods related tonon-VSV rhabdoviruses or pseudotyped rhabdoviruses and their use asanti-cancer therapeutics.

I. FAMILY RHABDOVIRIDAE (RHABDOVIRUS)

The archetypal rhabdoviruses are rabies and vesicular stomatitis virus(VSV), the most studied of this virus family. Although these virusesshare similar morphologies, they are very different in their life cycle,host range, and pathology. Rhabdovirus is a family of bullet shapedviruses having non-segmented (−)sense RNA genomes. There are greaterthan 250 Rhabdoviruses known that infect mammals, fish, insects, andplants. The family is split into at least 5 genera: (1) Lyssavirus:including Rabies virus, other mammalian viruses, some insect viruses;(2) Vesiculovirus: including Vesicular Stomatitis Virus (VSV); (3)Ephemerovirus: including Bovine ephemeral fever virus (vertebrates); (4)Cytorhabdovirus: including Lettuce necrotic yellows virus (plants); and(5) Nucleorhabdovirus: including Potato yellow dwarf virus (plants). Ithas also been suggested that there is a supergroup of rhabdovirusdenoted Dimarhabdovirus that include a variety of rhabdoviruses thatinfect both mammals and insects.

The family Rhabdovirus includes, but is not limited to: Arajas virus,Chandipura virus (AF128868/gi:4583436, AJ810083/gi:57833891,AY871800/gi:62861470, AY871799/gi:62861468, AY871798/gi:62861466,AY871797/gi:62861464, AY871796/gi:62861462, AY871795/gi:62861460,AY871794/gi:62861459, AY871793/gi:62861457, AY871792/gi:62861455,AY871791/gi:62861453), Cocal virus (AF045556/gi:2865658), Isfahan virus(AJ810084/gi:57834038), Maraba virus (SEQ ID NO:1-6), Carajas virus (SEQID NO:7-12, AY335185/gi:33578037), Piry virus (D26175/gi:442480,Z15093/gi:61405), Vesicular stomatitis Alagoas virus, BeAn 157575 virus,Boteke virus, Calchaqui virus, Eel virus American, Gray Lodge virus,Jurona virus, Klamath virus, Kwatta virus, La Joya virus, Malpais Springvirus, Mount Elgon bat virus (DQ457103/gi|91984805), Perinet virus(AY854652/gi:71842381), Tupaia virus (NC_007020/gi:66508427),Farmington, Bahia Grande virus (SEQ ID NO:13-18), Muir Springs virus,Reed Ranch virus, Hart Park virus, Flanders virus (AF523199/gi:25140635,AF523197/gi:25140634, AF523196/gi:25140633, AF523195/gi:25140632,AF523194/gi:25140631, AF1012179/gi:25140630), Kamese virus, Mosqueirovirus, Mossuril virus, Barur virus, Fukuoka virus(AY854651/gi:71842379), Kern Canyon virus, Nkolbisson virus, Le Dantecvirus (AY854650/gi:71842377), Keuraliba virus, Connecticut virus, NewMinto virus, Sawgrass virus, Chaco virus, Sena Madureira virus, Timbovirus, Almpiwar virus (AY854645/gi:71842367), Aruac virus, Bangoranvirus, Bimbo virus, Bivens Ann virus, Blue crab virus, Charlevillevirus, Coastal Plains virus, DakArK 7292 virus, Entamoeba virus, Garbavirus, Gossas virus, Humpty Doo virus (AY854643/gi:71842363), Joinjakakavirus, Kannamangalam virus, Kolongo virus (DQ457100/gi|91984799nucleoprotein (N) mRNA, partial cds); Koolpinyah virus, Kotonkon virus(DQ457099/gi|91984797, AY854638/gi:71842354); Landjia virus, Manitobavirus, Marco virus, Nasoule virus, Navarro virus, Ngaingan virus(AY854649/gi:71842375), Oak-Vale virus (AY854670/gi:71842417), Obodhiangvirus (DQ457098/gi|91984795), Oita virus (AB116386/gi:46020027), Ouangovirus, Parry Creek virus (AY854647/gi:71842371), Rio Grande cichlidvirus, Sandjimba virus (DQ457102/gi|91984803), Sigma virus(AH004209/gi:1680545, AH004208/gi:1680544, AH004206/gi:1680542), Sripurvirus, Sweetwater Branch virus, Tibrogargan virus(AY854646/gi:71842369), Xiburema virus, Yata virus, Rhode Island,Adelaide River virus (U10363/gi:600151, AF234998/gi:10443747,AF234534/gi:9971785, AY854635/gi:71842348), Berrimah virus(AY854636/gi:71842350]), Kimberley virus (AY854637/gi:71842352), orBovine ephemeral fever virus (NC_002526/gi:10086561).

Certain unassigned serotypes include (1) Bahia Grande group (BahiaGrande virus (BGV), Muir Springs virus (MSV), Reed Ranch virus (RRV);(2) Hart Park group (Flanders virus (FLAV), Hart Park virus (HPV),Kamese virus (KAMV), Mosqueiro virus (MQOV), Mossuril virus (MOSV); (3)Kern Canyon group (Barur virus (BARV), Fukuoka virus (FUKAV), KernCanyon virus (KCV), Nkolbisson virus (NKOV); (4) Le Dantec group (LeDantec virus (LDV), Keuraliba virus (KEUV), (5) Sawgrass group(Connecticut virus (CNTV), New Minto virus (NMV), Sawgrass virus (SAWV);(6) Timbo group (Chaco virus (CHOV), Sena Madureira virus (SMV), Timbovirus (TIMV); and (7) other unassigned viruses (Almpiwar virus (ALMV),Aruac virus (ARUV), Bangoran virus (BGNV), Bimbo virus (BBOV), BivensArm virus (SAV), Blue crab virus (BCV), Charleville virus (CHVV),Coastal Plains virus (CPV), DakArK 7292 virus (DAKV-7292), Entamoebavirus (ENTV), Garba virus (GARV), Gossas virus (GOSV), Humpty Doo virus(HDOOV), Joinjakaka virus (JOIV), Kannamangalam virus (KANV), Kolongovirus (KOLV), Koolpinyah virus (KOOLV), Kotonkon virus (KOTV), Landjiavirus (LJAV), Manitoba virus (MNTBV), Marco virus (MCOV), Ngaingan,Nasoule virus (NASV), Navarro virus (NAVV), Ngaingan virus (NGAV),Oak-Vale virus (OVRV), Obodhiang virus (OBOV), Oita virus (OITAV),Ouango virus (OUAV), Parry Creek virus (PCRV), Rio Grande cichlid virus(RGRCV), Sandjimba virus (SJAV), Sigma virus [X91062] (SIGMAV), Sripurvirus (SRIV), Sweetwater Branch virus (SWBV), Tibrogargan virus (TIBV),Xiburema virus (XIBV), Yata virus (YATAV).

Aspects of the invention may include, but is not limited to selectingnon-VSV rhabdovirus or pseudotyped rhabdovirus based on growth inmammalian cell lines, lack of or minimal toxicity in adult mice(animals), lack of or minimal toxicity in suckling mice (animals).

A. Rhabdoviral Genome

Typically the rhabdovirus genome is approximately 11-15 kb with anapproximately 50 nucleotide 3′ leader and an approximately 60 nucleotidenon-translated 5′ region of a (−) sense viral RNA (vRNA). Typically,rhabdovirus vRNA has 5 genes encoding 5 proteins. Rhabdoviruses have aconserved polyadenylation signal at the end of each gene and a shortintergenic region between each of the 5 genes. All Rhabdoviruses containfive genes which encode the nucleocapsid protein (N), Phosphoprotein (P,also designated NS), matrix protein (M), glycoprotein (G), and largeprotein (L). Typically these genes are ordered on negative sense vRNA asfollows: 3′-N-P-M-G-(X)-L-5′. The order of the genes is important as itdictates the proportion of proteins synthesized. Any manipulations of aRhabdovirus genome will typically include at least five transcriptiondomains to maintain ability to infect and replicate at high levels.Rhabdoviruses have an endogenous RNA polymerase for transcription ofplus sense messenger RNA (mRNA). The X gene does not occur in allRhabdoviruses. The X gene encodes a nonstructural protein found in thefish infectious hematopoietic necrosis virus (GenBankDQ164103/gi|76262981; DQ164102/gi|76262979; DQ164101/gi|76262977;DQ164100/gi|76262975; DQ164099/gi|76262973; AB250935/gi|112821165;AB250934/gi|112821163; AB250933/gi|112821161; AB250932/gi|112821159;AB250931/gi|112821157; AB250930/gi|112821155; AB250929/gi|1128211 53;AB250928/gi|112821151; AB250927/gi|112821149, describing the G proteinencoding nucleotide sequence), a nonstructural glycoprotein in thebovine ephemeral fever virus and a pseudogene in the rabies virus. Theextra (X) gene has been found in different locations on the Rhabdovirusgenome. Synthesis of the M protein in infected cells is cytopathic tothe cell, and will eventually result in cell death.

Transmission of rhabdovirus varies depending on virus/host, but most aretransmitted by direct contact—e.g., transmission of rabies by animalbites or insect vector. There is a long incubation period in vivo, butthis is not reflected in the kinetics of virus replication in culture.The G protein spikes bind to receptors on the surface of host cells andthe viruses enters the cell by endocytosis and fusion with the membraneof the vesicle, mediated by the G protein.

With no intent to be limited to a particular theory, the receptormolecules for rhabdoviruses are believed to be phospholipids rather thanspecific proteins. Rhabdoviral replication occurs in the cytoplasm—boththe L and NS proteins are necessary for transcription—neither functionalone. Five monocistronic mRNAs are produced, capped at the 5′ end andpolyadenylated at the 3′ end and each containing the leader sequencefrom the 3′ end of the vRNA at the 5′ end of the message. These mRNAsare made by sequential transcription of the ORFs in the virus genome andit has been shown that the intergenic sequence is responsible fortermination and re-initiation of transcription by the polymerase betweeneach gene, thus producing separate transcripts.

Progeny vRNA is made from a (+)sense intermediate. The genome isreplicated by the L+P polymerase complex (as in transcription), butadditional host cell factors are also required. It is characteristic ofRhabdoviruses that these events all occur in a portion of the cytoplasmwhich acts as a virus ‘factory’ and appears as a characteristiccytoplasmic inclusion body.

B. Viral Protein Variants

In certain embodiments, a rhabdovirus or a non-VSV rhabdovirus willcomprise a variant of one or more of the N, P, M, G, and/or L proteins.In certain aspects of the invention these viral protein variants can becomprised in a proteinaceous composition, which is further definedbelow. Proteinaceous compositions include viral particles and othercompositions having one or more viral protein components. Thesepolypeptide variant(s) can be engineered or selected for a modificationin one or more physiological or biological characteristics, such as hostcell range, host cell specificity, toxicity to non-target cells ororgans, replication, cytotoxicity to a target cell, killing of cancercells, stasis of cancer cells, infectivity, manufacturing parameters,size of virus particle, stability of viral particles, in vivo clearance,immunoreactivity, and the like. These polypeptide variant can beengineered by using a variety of methodology know in the art, includingvarious mutagenesis techniques described see below. In certain aspects,the N, P, M, G, and/or L proteins can be heterologous to a virus (e.g.,a VSV may comprise a Isfahan G protein or variant thereof).

C. Recombinant Rhabdoviruses

Recombinant rhabdovirus can be produced (1) entirely using cDNAs or (2)a combination of cDNAs transfected into a helper cell, or (3) cDNAstransfected into a cell, which is further infected with a minivirusproviding in trans the remaining components or activities needed toproduce either an infectious or non-infectious recombinant rhabdovirus.Using any of these methods (e.g., minivirus, helper cell line, or cDNAtransfection only), the minimum components required are an RNA moleculecontaining the cis-acting signals for (1) encapsidation of the genomic(or antigenomic) RNA by the Rhabdovirus N protein, and (2) replicationof a genomic or antigenomic (replicative intermediate) RNA equivalent.

By a replicating element or replicon, the inventors mean a strand of RNAminimally containing at the 5′ and 3′ ends the leader sequence and thetrailer sequence of a rhabdovirus. In the genomic sense, the leader isat the 3′ end and the trailer is at the 5′ end. Any RNA-placed betweenthese two replication signals will in turn be replicated. The leader andtrailer regions further must contain the minimal cis-acting elements forpurposes of encapsidation by the N protein and for polymerase bindingwhich are necessary to initiate transcription and replication.

For preparing engineered rhabdoviruses a minivirus containing the G genewould also contain a leader region, a trailer region and a G gene withthe appropriate initiation and termination signals for producing a Gprotein mRNA. If the minivirus further comprises a M gene, theappropriate initiation and termination signals for producing the Mprotein mRNA must also present.

For any gene contained within the engineered rhabdovirus genome, thegene would be flanked by the appropriate transcription initiation andtermination signals which will allow expression of those genes andproduction of the protein products. Particularly a heterologous gene,which is a gene that is typically not encoded by a rhabdovirus asisolated from nature or contains a rhabdovirus coding region in aposition, form or context that it typically is not found, e.g., achimeric G-protein.

To produce “non-infectious” engineered Rhabdovirus, the engineeredRhabdovirus must have the minimal replicon elements and the N, P, and Lproteins and it must contain the M gene (one example is the ΔG or G-lessconstruct, which is missing the coding region for the G protein). Thisproduces virus particles that are budded from the cell, but arenon-infectious particles. To produce “infectious” particles, the virusparticles must additionally comprise proteins that can mediate virusparticle binding and fusion, such as through the use of an attachmentprotein or receptor ligand. The native receptor ligand of rhabdovirusesis the G protein.

A “suitable cell” or “host cell” means any cell that would permitassembly of the recombinant rhabdovirus.

To prepare infectious virus particles, an appropriate cell line (e.g.,BHK cells) is first infected with vaccinia virus vTF7-3 (Fuerst et al.,1986) or equivalent which encodes a T7 RNA polymerase or other suitablebacteriophage polymerase such as the T3 or SP6 polymerases (see Usdin etal., 1993 or Rodriguez et al., 1990). The cells are then transfectedwith individual cDNA containing the genes encoding the G, N, P, L and MRhabdovirus proteins. These cDNAs will provide the proteins for buildinga recombinant Rhabdovirus particle. Cells can be transfected by anymethod known in the art (e.g., liposomes, electroporation, etc.).

Also transfected into the cell line is a “polycistronic cDNA” containingthe rhabdovirus genomic RNA equivalent. If the infectious, recombinantrhabdovirus particle is intended to be lytic in an infected cell, thenthe genes encoding for the N, P, M and L proteins must be present aswell as any heterologous nucleic acid segment. If the infectious,recombinant rhabdovirus particle is not intended to be lytic, then thegene encoding the M protein is not included in the polycistronic DNA. By“polycistronic cDNA” it is meant a cDNA comprising at leasttranscription units containing the genes which encode the N, P and Lproteins. The recombinant rhabdovirus polycistronic DNA may also containa gene encoding a protein variant or polypeptide fragment thereof, or atherapeutic nucleic acid. Alternatively, any protein to be initiallyassociated with the viral particle first produced or fragment thereofmay be supplied in trans.

Another embodiment contemplated is a polycistronic cDNA comprising agene encoding a reporter protein or fluorescent protein (e.g., greenfluorescent protein and its derivatives, β-galactosidase, alkalinephosphatase, luciferase, chloramphenicol acetyltransferase, etc.), theN-P-L or N-P-L-M genes, and/or a fusion protein or a therapeutic nucleicacid. Another polycistronic DNA contemplated may contain a gene encodinga protein variant, a gene encoding a reporter, a therapeutic nucleicacid, and/or either the N-P-L genes or the N-P-L-M genes.

The first step in generating a recombinant rhabdovirus is expression ofan RNA that is a genomic or antigenomic equivalent from a cDNA. Thenthat RNA is packaged by the N protein and then replicated by the P/Lproteins. The virus thus produced can be recovered. If the G protein isabsent from the recombinant RNA genome, then it is typically supplied intrans. If both the G and the M proteins are absent, then both aresupplied in trans.

For preparing “non-infectious rhabdovirus” particles, the procedure maybe the same as above, except that the polycistronic cDNA transfectedinto the cells would contain the N, P and L genes of the Rhabdovirusonly. The polycistronic cDNA of non-infectious rhabdovirus particles mayadditionally contain a gene encoding a reporter protein or a therapeuticnucleic acid. For additional description regarding methods of producinga recombinant rhabdovirus lacking the gene encoding the G protein, seeTakada et al. (1997).

1. Culturing of Cells to Produce Virus

Transfected cells are usually incubated for at least 24 hr at thedesired temperature, usually about 37° C. For non-infectious virusparticles, the supernatant is collected and the virus particlesisolated. For infectious virus particles, the supernatant containingvirus is harvested and transferred to fresh cells. The fresh cells areincubated for approximately 48 hours, and the supernatant is collected.

2. Purification of the Recombinant Rhabdovirus

The terms “isolation” or “isolating” a Rhabdovirus means the process ofculturing and purifying the virus particles such that very littlecellular debris remains. One example would be to take the virioncontaining supernatant and pass them through a 0.1-0.2 micron pore sizefilter (e.g., Millex-GS, Millipore) to remove the virus and cellulardebris. Alternatively, virions can be purified using a gradient, such asa sucrose gradient. Recombinant rhabdovirus particles can then bepelleted and resuspended in whatever excipient or carrier is desired.Titers can be determined by indirect immunofluorescence using antibodiesspecific for particular proteins.

3. Methods of Making Recombinant Rhabdoviruses Using cDNAs and aMinivirus or a Helper Cell Line

Both “miniviruses” and “helper cells” (also known as “helper celllines”) provide the same thing: to provide a source of rhabdovirusproteins for rhabdovirus virion assembly. One example of a rhabdovirusminivirus is the VSV minivirus which expresses only the G and M protein,as reported by Stillman et al., (1995). Helper viruses and minivirusesare used as methods of providing rhabdovirus proteins that are notproduced from transfected DNA encoding the genes for rhabdovirusproteins.

When using a minivirus, cells are infected with vaccinia virus asdescribed above for purposes of providing T7 RNA polymerase. The desiredpolycistronic RNA, and plasmids containing the N, P and L genes aretransfected into cells. The transfection mix is removed afterapproximately 3 hrs, and cells are infected with the minivirus at amultiplicity of infection (m.o.i.) of about 1. The minivirus suppliesthe missing G and/or M proteins. The polycistronic RNA transfected intothe cell will depend on whether an infectious or non-infectiousrecombinant rhabdovirus is wanted.

Alternatively, a minivirus could be used to provide the N, P, and Lgenes. The minivirus could also be used to produce the M protein inaddition to N, P, and L. The minivirus also can produce the G protein.

When using a helper cell line, the genes encoding the missingrhabdovirus proteins are produced by the helper cell line. The helpercell line has N, P, L, and G proteins for production of recombinantrhabdovirus particles which does not encode wild-type G protein. Theproteins are expressed from genes or DNAs that are not part of therecombinant virus genome. These plasmids or other vector system isstably incorporated into the genome of the cell line. The proteins arethen produced from the cell's genome and not from a replicon in thecytoplasm. The helper cell line can then be transfected with apolycistronic DNA and plasmid cDNAs containing the other rhabdovirusgenes not expressed by the helper virus. The polycistronic RNA used willdepend on whether an infectious or non-infectious recombinantrhabdovirus is desired. Otherwise, supply of missing gene products(e.g., G and/or M) would be accomplished as described above.

II. VIRAL COMPOSITIONS

The present invention concerns rhabdoviruses that are advantageous inthe study and treatment of hyperproliferative or neoplastic cells (e.g.,cancer cells) and hyperproliferative or neoplastic conditions (e.g.,cancer) in a patient. It may concern, but is not limited to,rhabdoviruses with a reduced neurovirulence, e.g., non-VSVrhabdoviruses. In certain aspects rhabdovirus that encode or contain oneor more protein components (N, P, M, G, and/or L proteins) or a nucleicacid genome distinct from those of VSV (i.e., at least or at most 10,20, 40, 50, 60, 70, 80% identical at the amino acid or nucleotidelevel), and/or that have been constructed with one or more mutations orvariations as compared to a wild-type virus or viral proteins such thatthe virus has desirable properties for use against cancer cells, whilebeing less toxic or non-toxic to non-cancer cells than the virus asoriginally isolated or VSV. The teachings described below providevarious examples of protocols for implementing methods and compositionsof the invention. They provide background for generating mutated orvariant viruses through the use of bioselection or recombinant DNA ornucleic acid technology.

A. Proteinaceous Compositions

Proteinaceous compositions of the invention include viral particles andcompositions including the viral particles, as well as isolatedpolypeptides. In certain embodiments, the present invention concernsgenerating or isolating pseudotyped or non-VSV oncolytic rhabdoviruses(rhabdoviruses that lyse, kill, or retard growth of cancer cells). Incertain embodiments, rhabdoviruses will be engineered to includepolypeptide variants of rhabdovirus proteins (N, P, M, G, and/or L)and/or therapeutic nucleic acids that encode therapeutic polypeptides.Other aspects of the invention include the isolation of rhabdovirusesthat lack one or more functional polypeptides or proteins. In otherembodiments, the present invention concerns rhabdoviruses and their usein combination with or included within proteinaceous compositions aspart of a pharmaceutically acceptable formulation.

As used herein, a “protein” or “polypeptide” refers to a moleculecomprising polymer of amino acid residues. In some embodiments, awild-type version of a protein or polypeptide are employed, however, inmany embodiments of the invention, all or part of a viral protein orpolypeptide is absent or altered so as to render the virus more usefulfor the treatment of a patient. The terms described above may be usedinterchangeably herein. A “modified protein” or “modified polypeptide”or “variant protein” or “variant polypeptide” refers to a protein orpolypeptide whose chemical structure or amino acid sequence is alteredwith respect to the wild-type or a reference protein or polypeptide. Insome embodiments, a modified protein or polypeptide has at least onemodified activity or function (recognizing that proteins or polypeptidesmay have multiple activities or functions). The modified activity orfunction may be reduced, diminished, eliminated, enhanced, improved, oraltered in some other way (such as infection specificity) with respectto that activity or function in a wild-type protein or polypeptide, orthe characteristics of virus containing such a polypeptide. It iscontemplated that a modified protein or polypeptide may be altered withrespect to one activity or function yet retain wild-type or unalteredactivity or function in other respects. Alternatively, a modifiedprotein may be completely nonfunctional or its cognate nucleic acidsequence may have been altered so that the polypeptide is no longerexpressed at all, is truncated, or expresses a different amino acidsequence as a result of a frameshift or other modification.

In certain embodiments the size of a recombinant protein or polypeptidemay comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650,675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000,1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 or greater aminomolecule residues, and any range derivable therein. It is contemplatedthat polypeptides may be modified by truncation, rendering them shorterthan their corresponding unaltered form or by fusion or domain shufflingwhich may render the altered protein longer.

As used herein, an “amino molecule” refers to any amino acid, amino acidderivative, or amino acid mimic as would be known to one of ordinaryskill in the art. In certain embodiments, the residues of theproteinaceous molecule are sequential, without any non-amino moleculeinterrupting the sequence of amino molecule residues. In otherembodiments, the sequence may comprise one or more non-amino moleculemoieties. In particular embodiments, the sequence of residues of theproteinaceous molecule may be interrupted by one or more non-aminomolecule moieties. Accordingly, the term “proteinaceous composition”encompasses amino molecule sequences comprising at least one of the 20common amino acids in naturally synthesized proteins, or at least onemodified or unusual amino acid.

Proteinaceous compositions may be made by any technique known to thoseof skill in the art, including the expression of proteins, polypeptides,or peptides through standard molecular biological techniques, theisolation of proteinaceous compounds from natural sources, or thechemical synthesis of proteinaceous materials. The nucleotide andpolypeptide sequences for various rhabdovirus genes or genomes have beenpreviously disclosed, and may be found at computerized databases knownto those of ordinary skill in the art. One such database is the NationalCenter for Biotechnology Information's GenBank and GenPept databases,which can be accessed via the internet at ncbi.nlm.nih.gov/. The codingregions for these known genes and viruses may be amplified and/orexpressed using the techniques disclosed herein or as would be know tothose of ordinary skill in the art.

B. Functional Aspects

When the present application refers to the function or activity of viralproteins or polypeptides, it is meant to refer to the activity orfunction of that viral protein or polypeptide under physiologicalconditions, unless otherwise specified. For example, the G protein isinvolved in specificity and efficiency of binding and infection ofparticular cell types. Determination of which molecules possess thisactivity may be achieved using assays familiar to those of skill in theart, such as infectivity assays, protein binding assays, plaque assaysand the like.

C. Variants of Viral Polypeptides

Amino acid sequence variants of the polypeptides of the presentinvention can be substitutional, insertional or deletion variants. Amutation in a gene encoding a viral polypeptide may affect 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475,500 or more non-contiguous or contiguous amino acids (i.e., segment) ofa polypeptide, as compared to a wild-type or unaltered polypeptide orother reference polypeptide. Various polypeptides encoded byrhabdoviruses may be identified by reference to GenBank AccessionNumbers and the related public database entries for each of the virusesdisclosed herein, all GenBank entries related to the familyrhabdoviridae are incorporated herein by reference.

Deletion variants lack one or more residues of the native, unaltered orwild-type protein. Individual residues can be deleted, or all or part ofa domain (such as a catalytic or binding domain) can be deleted. A stopcodon may be introduced (by substitution or insertion) into an encodingnucleic acid sequence to generate a truncated protein. Insertionalmutants typically involve the addition of material at a non-terminalpoint in the polypeptide, a specific type of insert is a chimericpolypeptide that include homologous or similar portions of a relatedprotein in place of the related portion of a target protein. This mayinclude the insertion of an immunoreactive epitope or simply one or moreresidues. Terminal additions, typically called fusion proteins, may alsobe generated.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, with or withoutthe loss of other functions or properties. Substitutions may beconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine. Alternatively, substitutions may benon-conservative such that a function or activity of the polypeptide isaffected. Non-conservative changes typically involve substituting aresidue with one that is chemically dissimilar, such as a polar orcharged amino acid for a nonpolar or uncharged amino acid, and viceversa.

The term “functionally equivalent codon” is used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine or serine, and also refers to codons that encode biologicallyequivalent amino acids (see Table 1, below).

TABLE 1 Codon Table Amino Acids Codons Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Scrine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setas forth herein, including having a certain biological activity. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

The following is a discussion based upon changing of the amino acids ofa N, P, L, or G protein to create an equivalent, or even an improved,molecule. For example, certain amino acids may be substituted for otheramino acids in a protein structure without appreciable loss ofinteractive binding capacity with structures such as, for example,antigen-binding regions of antibodies or binding sites on substratemolecules. Since it is the interactive capacity and nature of a proteinthat defines that protein's biological functional activity, certainamino acid substitutions can be made in a protein sequence, and in itsunderlying DNA coding sequence, and nevertheless produce a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the DNA sequences of rhabdovirus withoutappreciable loss of biological utility or activity of interest, asdiscussed below.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring a biologic function on a protein is generally understood inthe art (Kyte and Doolittle, 1982). It is accepted that the relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (0.5); histidine*−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (2.3); phenylalanine (−2.5); tryptophan(−3.4). It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still produce abiologically equivalent and immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those that are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions generally are based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take into consideration the variousforegoing characteristics are well known to those of skill in the artand include: arginine and lysine; glutamate and aspartate; serine andthreonine; glutamine and asparagine; and valine, leucine and isoleucine.

III. NUCLEIC ACID MOLECULES

The present invention includes polynucleotides isolatable from cellsthat are capable of expressing all or part of a viral protein orpolypeptide. In some embodiments of the invention, it concerns all orparts of a viral genome that has been specifically mutated or altered togenerate a virus or viral polypeptide, e.g., a pseudotyped or non-VSVrhabdoviral polypeptide or virus, with certain properties and/orcharacteristics. The polynucleotides may encode a peptide or polypeptidecontaining all or part of a viral or heterologous amino acid sequence orbe engineered so they do not encode such a viral polypeptide or encode aviral polypeptide having at least one function or activity added,increased, reduced, added, diminished, or absent. Recombinant proteinscan be purified from expressing cells to yield active proteins. Thegenome of rhabdovirus members may be found in GenBank Accession Numbersin the NCBI database or similar databases, each of which is incorporatedherein by reference.

A. Polynucleotides Encoding Native or Modified Proteins

As used herein, the term “RNA, DNA, or nucleic acid segment” refers to aRNA, DNA, or nucleic acid molecule that has been isolated free of totalgenomic DNA or other contaminants. Therefore, a nucleic acid segmentencoding a polypeptide refers to a nucleic acid segment that containswild-type, polymorphic, or mutant polypeptide-coding sequences yet isisolated away from, or purified free from, genomic nucleic acid(s).Included within the term “nucleic acid segment” are polynucleotides,nucleic acid segments smaller than a polynucleotide, and recombinantvectors, including, for example, plasmids, cosmids, phage, viruses, andthe like.

As used in this application, the term “rhabdovirus polynucleotide” canrefer to pseudotyped or non-VSV rhabdoviral nucleic acid moleculeencoding at least one non-VSV rhabdovirus polypeptide. In certainembodiments the polynucleotide has been isolated free of other nucleicacids. Similarly, a “Maraba virus, Carajas virus, Muir Springs virusand/or Bahia Grande virus polynucleotide” refers to a nucleic acidmolecule encoding a Maraba virus, Carajas virus, Muir Springs virusand/or Bahia Grande virus polypeptide that has been isolated from othernucleic acids. A “rhabdovirus genome” or a “Maraba virus, Carajas virus,Muir Springs virus and/or Bahia Grande virus genome” refers to a VSV ora non-VSV nucleic acid molecule that can be provided to a host cell toyield a viral particle, in the presence or absence of a helper virus orcomplementing coding regions supplying other factors in trans. Thegenome may or may have not been recombinantly mutated as compared towild-type or an unaltered virus.

The term “cDNA” is intended to refer to DNA prepared using RNA as atemplate. There may be times when the full or partial genomic sequenceis preferred.

It also is contemplated that a particular polypeptide from a givenspecies may be represented by natural variants that have slightlydifferent nucleic acid sequences but, nonetheless, encode the sameprotein (see Table 1 above).

Similarly, a polynucleotide encoding an isolated or purified wild-type,or modified polypeptide refers to a DNA segment including wild-type ormutant polypeptide coding sequences and, in certain aspects, regulatorysequences, isolated substantially away from other naturally occurringgenes or protein encoding sequences. In this respect, the term “gene” isused for simplicity to refer to a nucleic acid unit encoding a protein,polypeptide, or peptide (including any sequences required for propertranscription, post-translational modification, or localization). Aswill be understood by those in the art, this functional term includesgenomic sequences, cDNA sequences, and smaller engineered nucleic acidsegments that express, or may be adapted to express, proteins,polypeptides, domains, peptides, fusion proteins, and mutants. A nucleicacid encoding all or part of a native or modified polypeptide maycontain a contiguous nucleic acid of: 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490,500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630,640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770,780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910,920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040,1050, 1060, 1070, 1080, 1090, 1095, 1100, 1500, 2000, 2500, 3000, 3500,4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000, ormore nucleotides, nucleosides, or base pairs.

In particular embodiments, the invention concerns isolated nucleic acidsegments and recombinant vectors incorporating nucleic acid sequencesthat encode a wild-type or mutant rhabdovirus polypeptide(s) thatincludes within its amino acid sequence a contiguous amino acid sequencein accordance with, or essentially corresponding to a nativepolypeptide. The term “recombinant” may be used in conjunction with apolypeptide or the name of a specific polypeptide, and this generallyrefers to a polypeptide produced from a nucleic acid molecule that hasbeen manipulated in vitro or that is the replicated product of such amolecule.

In other embodiments, the invention concerns isolated nucleic acidsegments and recombinant vectors incorporating nucleic sequences thatencode a polypeptide or peptide that includes within its amino acidsequence a contiguous amino acid sequence in accordance with, oressentially corresponding to one or more rhabdovirus polypeptide.

The nucleic acid segments used in the present invention, regardless ofthe length of the coding sequence itself, may be combined with othernucleic acid sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant nucleic acid protocol.

It is contemplated that the nucleic acid constructs of the presentinvention may encode hill-length polypeptide(s) from any source orencode a truncated or modified version of the polypeptide(s), forexample a truncated rhabdovirus polypeptide, such that the transcript ofthe coding region represents the truncated version. The truncatedtranscript may then be translated into a truncated protein.Alternatively, a nucleic acid sequence may encode a full-lengthpolypeptide sequence with additional heterologous coding sequences, forexample to allow for purification of the polypeptide, transport,secretion, post-translational modification, or for therapeutic benefitssuch as targeting or efficacy. As discussed above, a tag or otherheterologous polypeptide may be added to the modifiedpolypeptide-encoding sequence, wherein “heterologous” refers to apolypeptide or segment thereof that is not the same as the modifiedpolypeptide or found associated with or encoded by the naturallyoccurring virus.

In a non-limiting example, one or more nucleic acid construct may beprepared that include a contiguous stretch of nucleotides identical toor complementary to a particular viral segment, such as a rhabdovirus N,P, M, G, or L gene. A nucleic acid construct may be at least 20, 30, 40,50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000,5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 30,000,50,000, 100,000, 250,000, 500,000, 750,000, to at least 1,000,000nucleotides in length, as well as constructs of greater size, up to andincluding chromosomal sizes (including all intermediate lengths andintermediate ranges). It will be readily understood that “intermediatelengths” and “intermediate ranges,” as used herein, means any length orrange including or between the quoted values (i.e., all integersincluding and between such values).

The nucleic acid segments used in the present invention encompassmodified nucleic acids that encode modified polypeptides. Such sequencesmay arise as a consequence of codon redundancy and functionalequivalency that are known to occur naturally within nucleic acidsequences and the proteins thus encoded. Alternatively, functionallyequivalent proteins or peptides may be created via the application ofrecombinant DNA technology, in which changes in the protein structuremay be engineered, based on considerations of the properties of theamino acids being exchanged. Changes designed by human may be introducedthrough the application of site-directed mutagenesis techniques, e.g.,to introduce improvements to the antigenicity or lack thereof of theprotein, to reduce toxicity effects of the protein in vivo to a subjectgiven the protein, or to increase the efficacy of any treatmentinvolving the protein or a virus comprising such protein.

In certain other embodiments, the invention concerns isolated nucleicacid segments and recombinant vectors that include within their sequencea contiguous nucleic acid sequence from that shown in sequencesidentified herein (and/or incorporated by reference). Such sequences,however, may be mutated to yield a protein product whose activity isaltered with respect to wild-type.

It also will be understood that this invention is not limited to theparticular nucleic acid and amino acid sequences of these identifiedsequences. Recombinant vectors and isolated nucleic acid segments maytherefore variously include rhabdovirus-coding regions themselves,coding regions bearing selected alterations or modifications in thebasic coding region, or they may encode larger polypeptides thatnevertheless include rhabdovirus-coding regions, or may encodebiologically functional equivalent proteins or peptides that havevariant amino acids sequences.

The nucleic acid segments of the present invention can encoderhabdovirus proteins and peptides that are the biological functionalequivalent of, or variants or mutants of rhabdovirus that increase thetherapeutic benefit of the virus. Such sequences may arise as aconsequence of codon redundancy and functional equivalency that areknown to occur naturally within nucleic acid sequences and the proteinsthus encoded. Alternatively, functionally equivalent proteins orpeptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site directed mutagenesis techniques, e.g., to introduceimprovements in cancer cell binding of a viral protein.

B. Mutagenesis of Rhabdovirus Polynucleotides

In various embodiments, the rhabdovirus polynucleotide may be altered ormutagenized. Alterations or mutations may include insertions, deletions,point mutations, inversions, and the like and may result in themodulation, activation and/or inactivation of certain proteins ormolecular mechanisms, as well as altering the function, location, orexpression of a gene product, in particular rendering a gene productnon-functional. Where employed, mutagenesis of a polynucleotide encodingall or part of a rhabdovirus may be accomplished by a variety ofstandard, mutagenic procedures (Sambrook et al., 2001). Mutation is theprocess whereby changes occur in the quantity or structure of anorganism. Mutation can involve modification of the nucleotide sequenceof a single gene, blocks of genes or whole genomes. Changes in singlegenes may be the consequence of point mutations which involve theremoval, addition or substitution of a single nucleotide base within aDNA sequence, or they may be the consequence of changes involving theinsertion or deletion of large numbers of nucleotides.

1. Random Mutagenesis

a. Insertional Mutagenesis

Insertional mutagenesis is based on the inactivation of a gene viainsertion of a known nucleic acid fragment. Because it involves theinsertion of some type of nucleic acid fragment, the mutations generatedare generally loss-of-function, rather than gain-of-function mutations.However, there are several examples of insertions generatinggain-of-function mutations. Insertional mutagenesis may be accomplishedusing standard molecular biology techniques.

b. Chemical Mutagenesis

Chemical mutagenesis offers certain advantages, such as the ability tofind a full range of mutations with degrees of phenotypic severity, andis facile and inexpensive to perform. The majority of chemicalcarcinogens produce mutations in DNA. Benzo[a]pyrene, N-acetoxy-2-acetylaminofluorene and aflotoxin B1 cause GC to TA transversions in bacteriaand mammalian cells. Benzo[a]pyrene also can produce base substitutionssuch as AT to TA. N-nitroso compounds produce GC to AT transitions.Alkylation of the O4 position of thymine induced by exposure ton-nitrosourea results in TA to CC transitions.

c. Radiation Mutagenesis

Biological molecules are degraded by ionizing radiation. Adsorption ofthe incident energy leads to the formation of ions and free radicals,and breakage of some covalent bonds. Susceptibility to radiation damageappears quite variable between molecules, and between differentcrystalline forms of the same molecule. It depends on the totalaccumulated dose, and also on the dose rate (as once free radicals arepresent, the molecular damage they cause depends on their naturaldiffusion rate and thus upon real time). Damage is reduced andcontrolled by making the sample as cold as possible. Ionizing radiationcauses DNA damage, generally proportional to the dose rate.

In the present invention, the term “ionizing radiation” means radiationcomprising particles or photons that have sufficient energy or canproduce sufficient energy to produce ionization (gain or loss ofelectrons). An exemplary and preferred ionizing radiation is anx-radiation. The amount of ionizing radiation needed in a given cell orfor a particular molecule generally depends upon the nature of that cellor molecule and the nature of the mutation target. Means for determiningan effective amount of radiation are well known in the art.

d. In Vitro Scanning Mutagenesis

Random mutagenesis also may be introduced using error prone PCR. Therate of mutagenesis may be increased by performing PCR in multiple tubeswith dilutions of templates. One particularly useful mutagenesistechnique is alanine scanning mutagenesis in which a number of residuesare substituted individually with the amino acid alanine so that theeffects of losing side-chain interactions can be determined, whileminimizing the risk of large-scale perturbations in protein conformation(Cunningham el al., 1989).

In vitro scanning saturation mutagenesis provides a rapid method forobtaining a large amount of stricture-function information including:(i) identification of residues that modulate ligand binding specificity,(ii) a better understanding of ligand binding based on theidentification of those amino acids that retain activity and those thatabolish activity at a given location, (iii) an evaluation of the overallplasticity of an active site or protein subdomain, (iv) identificationof amino acid substitutions that result in increased binding.

Site-Directed Mutagenesis

Structure-guided site-specific mutagenesis represents a powerful toolfor the dissection and engineering of protein-ligand interactions(Wells, 1996; Braisted et al., 1996). The technique provides for thepreparation and testing of sequence variants by introducing one or morenucleotide sequence changes into a selected DNA.

C. Vectors

To generate mutations in a rhabdovirus genome, native and modifiedpolypeptides may be encoded by a nucleic acid molecule comprised in avector. The term “vector” is used to refer to a carrier nucleic acidmolecule into which an exogenous nucleic acid sequence can be insertedfor introduction into a cell where it can be replicated. A nucleic acidsequence can be “exogenous,” which means that it is foreign to the cellinto which the vector is being introduced or that the sequence ishomologous to a sequence in the cell but in a position within the hostcell nucleic acid in which the sequence is ordinarily not found. Vectorsinclude plasmids, cosmids, viruses (bacteriophage, animal viruses, andplant viruses), and artificial chromosomes (e.g., YACs). One of skill inthe art would be well equipped to construct a vector through standardrecombinant techniques, which are described in Sambrook et al. (2001)and Ausubel et al. (1994), both incorporated herein by reference.

In addition to encoding a modified polypeptide such as modified Nprotein, P protein, M protein, G protein, or L protein, a vector mayencode non-modified polypeptide sequences such as a tag or targetingmolecule. Useful vectors encoding such fusion proteins include pINvectors (Inouye et al., 1985), vectors encoding a stretch of histidines,and pGEX vectors, for use in generating glutathione S-transferase (GST)soluble fusion proteins for later purification and separation orcleavage. A targeting molecule is one that directs the modifiedpolypeptide to a particular organ, tissue, cell, or other location in asubject's body. Alternatively, the targeting molecule alters the tropismof an organism, such as rhabdovirus for certain cell types, e.g., cancercells.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are translated into aprotein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

1. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements that bind regulatory proteins andmolecules, such as RNA polymerase and other transcription factors. Thephrases “operatively positioned,” “operatively coupled,” “operativelylinked,” “under control,” and “under transcriptional control” mean thata promoter is in a correct functional location and/or orientation inrelation to a nucleic acid sequence to control transcriptionalinitiation and/or expression of that sequence. A promoter may or may notbe used in conjunction with an “enhancer,” which refers to a cis-actingregulatory sequence involved in the transcriptional activation of anucleic acid sequence.

A promoter may be one naturally associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other prokaryotic, viral, or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression.

In addition to producing nucleic acid sequences of promoters andenhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202, 5,928,906, each incorporated herein by reference).Furthermore, it is contemplated the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

Naturally, it may be important to employ a promoter and/or enhancer thateffectively directs the expression of the DNA segment in the cell type,organelle, and organism chosen for expression. Those of skill in the artof molecular biology generally know the use of promoters, enhancers, andcell type combinations for protein expression, for example, see Sambrooket al. (2001), incorporated herein by reference. The promoters employedmay be constitutive, tissue-specific, cell selective (i.e., more activein one cell type as compared to another), inducible, and/or useful underthe appropriate conditions to direct high level expression of theintroduced nucleic acid segment, such as is advantageous in thelarge-scale production of recombinant proteins and/or peptides. Thepromoter may be heterologous or endogenous.

Several elements/promoters that may be employed, in the context of thepresent invention, to regulate the expression of a gene. This list isnot intended to be exhaustive of all the possible elements involved inthe promotion of expression but, merely, to be exemplary thereof. Alsoprovided are examples of inducible elements, which are regions of anucleic acid sequence that can be activated in response to a specificstimulus. Promoter/Enhancer (References) include: Immunoglobulin HeavyChain (Banerji et al., 1983; Gilles et al., 1983; Grosschedl et al.,1985; Atchinson et al., 1986, 1987; Imler et al., 1987; Weinberger etal., 1984; Kiledjian et al., 1988; Porton et al.; 1990); ImmunoglobulinLight Chain (Queen et al., 1983; Picard et al., 1984); T Cell Receptor(Luria et al., 1987; Winoto et al., 1989; Redondo et al.; 1990); HLA DQα and/or DQ β (Sullivan et al., 1987); β Interferon (Goodbourn et al.,1986; Fujita et al., 1987; Goodbourn et al., 1988); Interleukin-2(Greene et al., 1989); Interleukin-2 Receptor (Greene et al., 1989; Linet al., 1990); MHC Class II 5 (Koch et al., 1989); MHC Class II HLA-DRα(Sherman el al., 1989); β-Actin (Kawamoto et al., 1988; Ng et al.;1989); Muscle Creatine Kinase (MCK) (Jaynes et al., 1988; Horlick etal., 1989; Johnson et al., 1989); Prealbumin (Transthyretin) (Costa etal., 1988); Elastase I (Omitz et al., 1987); Metallothionein (MTII)(Karin et al., 1987; Culotta et al., 1989); Collagenase (Pinkert et at.,1987; Angel et al., 1987); Albumin (Pinkert et al., 1987; Tronche etal., 1989, 1990); α-Fetoprotein (Godbout et al., 1988; Campere et al.,1989); γ-Globin (Bodine at al., 1987; Perez-Stable et al., 1990);β-Globin (Trudel et al., 1987); c-fos (Cohen et al., 1987); c-HA-ras(Triesman, 1986; Deschamps et al., 1985); Insulin (Edlund et al., 1985);Neural Cell Adhesion Molecule (NCAM) (Hirsh et al., 1990);α1-Antitrypain (Latimer et al., 1990); H2B (TH2B) Histone (Hwang et al.,1990); Mouse and/or Type I Collagen (Ripe et al., 1989);Glucose-Regulated Proteins (GRP94 and GRP78) (Chang et al., 1989); RatGrowth Hormone (Larsen et al., 1986); Human Serum Amyloid A (SAA)(Edbrooke et al., 1989); Troponin I (TN I) (Yutzey et al., 1989);Platelet-Derived Growth Factor (PDGF) (Pech et al., 1989); DuchenneMuscular Dystrophy (Klamut et al., 1990); SV40 (Banerji et al., 1981;Moreau et al., 1981; Sleigh et al., 1985; Firak et al., 1986; Herr etal., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wang et al., 1986;Ondek et al., 1987; Kuhl et al., 1987; Schaffner et at., 1988); Polyoma(Swartzendruber et al., 1975; Vasseur et al., 1980; Katinka et al.,1980, 1981; Tyndell et al., 1981; Dandolo et at., 1983; de Villiers etal., 1984; Hen et al., 1986; Satake et al., 1988; Campbell et al.,1988); Retroviruses (Kriegler et al., 1982, 1983; Levinson et al., 1982;Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986; Miksicek etal., 1986; Celander et al., 1987; Thiesen et al., 1988; Celander et al.,1988; Chol et al., 1988; Reisman et al., 1989); Papilloma Virus (Campoet al., 1983; Lusky et al., 1983; Spandidos and Wilkie, 1983; Spalholzet al., 1985; Lusky et al., 1986; Cripe et al., 1987; Gloss et al.,1987; Hirochika et al., 1987; Stephens et al., 1987); Hepatitis B Virus(Bulla et al., 1986; Jameel et al., 1986; Shaul et al., 1987; Spandau etal., 1988; Vannice et al., 1988); Human Immunodeficiency Virus (Muesinget al., 1987; Hauber et al., 1988; Jakobovits et al., 1988; Feng at al.,1988; Takebe et al, 1988; Rosen et al., 1988; Berkhout et al., 1989;Laspia et al., 1989; Sharp et al., 1989; Braddock et al., 1989);Cytomegalovirus (CMV) (Weber et al., 1984; Boshart et al., 1985;Foecking et al., 1986); and Gibbon Ape Leukemia Virus (Holbrook et al.,1987; Quinn et al., 1989).

Inducible Elements (Element/Inducer (References)) include: MT II/PhorbolEster (TFA), Heavy metals (Palmiter et al., 1982; Haslinger et al.,1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987,Karin et al., 1987; Angel et al., 1987b; McNeall et al., 1989); MMTV(mouse mammary tumor virus)/Glucocorticoids (Huang et al., 1981; Lee etal., 1981; Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984;Ponta et al., 1985; Sakai et al., 1988); β-Interferon/poly(rI)x,poly(rc) (Tavernier et al., 1983); Adenovirus 5 E2/E1A (Imperiale etal., 1984); Collagenase/Phorbol Ester (TPA) (Angel et al., 1987a);Stromelysin/Phorbol Ester (TPA) (Angel et al., 1987b); SV40/PhorbolEster (TPA) (Angel et al., 1987b); Murine MX Gene/Interferon, NewcastleDisease Virus (Hug et al., 1988); GRP78 Gene/A23187 (Resendez et al.,1988); α-2-Macroglobulin/IL-6 (Kunz et al., 1989); Vimentin/Serum(Riffling et al., 1989); MHC Class I Gene H-2κb/Interferon (Blanar etal., 1989); HSP70/E1A, SV40 Large T Antigen (Taylor et al., 1989, 1990a,1990b); Proliferin/Phorbol Ester-TPA (Mordacq et al., 1989); TumorNecrosis Factor/PMA (Hensel et al., 1989); and Thyroid StimulatingHormone α Gene/Thyroid Hormone (Chatterjee et al., 1989).

The identity of tissue-specific or tissue-selective (i.e., promotersthat have a greater activity in one cell as compared to another)promoters or elements, as well as assays to characterize their activity,is well known to those of skill in the art. Examples of such regionsinclude the human LIMK2 gene (Nomoto et al. 1999), the somatostatinreceptor 2 gene (Kraus et al., 1998), murine epididymal retinoicacid-binding gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al.,1998), mouse alpha2 (XI) collagen (Tsumaki, et al., 1998), D1A dopaminereceptor gene (Lee, et al., 1997), insulin-like growth factor II (Wu etal., 1997), human platelet endothelial cell adhesion molecule-1(Almendro et al., 1996), and the SM22α promoter.

Additional viral promoters, cellular promoters/enhancers and induciblepromoters/enhancers that could be used in combination with the presentinvention are listed herein. Additionally any promoter/enhancercombination (as per the Eukaryotic Promoter Data Base EPDB) could alsobe used to drive expression of structural genes encoding oligosaccharideprocessing enzymes, protein folding accessory proteins, selectablemarker proteins or a heterologous protein of interest. Alternatively, atissue-specific promoter for cancer gene therapy (Table 2) or thetargeting of tumors (Table 3) may be employed with the nucleic acidmolecules of the present invention.

TABLE 2 Candidate Tissue-Specific Promoters for Cancer Gene TherapyTissue-specific Cancers in which Normal cells in which promoter promoteris active promoter is active Carcinoembryonic Most colorectal Colonicmucosa; antigen (CEA)* carcinomas; 50% of lung gastric mucosa; lungcarcinomas; 40-50% of epithelia; eccrine gastric carcinomas; most sweatglands; cells in pancreatic carcinomas; testes many breast carcinomasProstate-specific Most prostate carcinomas Prostate epithelium antigen(PSA) Vasoactive Majority of non-small cell Neurons; lymphocytes;intestinal peptide lung cancers mast cells; eosinophils (VIP) Surfactantprotein Many lung Type II pneumocytes; A (SP-A) adenocarcinomas cellsClara Human achaete- Most small cell lung Neuroendocrinc cells in scutehomolog cancers lung (hASH) Mucin-1 Most adenocarcinomas Glandularepithelial (MUC1)** (originating from any cells in breast and in tissue)respiratory, gastrointestinal, and genitourinary tractsAlpha-fetoprotein Most hepatocellular Hepatocytes (under carcinomas;possibly many certain conditions); testicular cancers testis AlbuminMost hepatocellular Hepatocytcs carcinomas Tyrosinase Most melanomasMelanocytes; astrocytes; Schwann cells; some neurons Tyrosine-bindingMost melanomas Melanocytes; protein (TRP) astrocytes, Schwann cells;some neurons Keratin 14 Presumably many Keratinocytes squamous cellcarcinomas (e.g.: Head and neck cancers) EBV LD-2 Many squamous cellKeratinocytes of upper carcinomas of head and digestive Keratinocytesneck of upper digestive tract Glial fibrillary Many astrocytomasAstrocytes acidic protein (GFAP) Myelin basic Many gliomasOligodendrocytes protein (MBP) Testis-specific Possibly many testicularSpermatazoa angiotensin- cancers converting enzyme (Testis-specific ACE)Osteocalcin Possibly many Osteoblasts osteosarcomas

TABLE 3 Candidate Promoters for Use with a Tissue-Specific Targeting ofTumors Cancers in which Normal cells in which Promoter Promoter isactive Promoter is active E2F-regulated Almost all cancers Proliferatingcells promoter HLA-G Many colorectal Lymphocytes; carcinomas; manymonocytes; melanomas; possibly spermatocytes; many other cancerstrophoblast FasL Most melanomas; many Activated leukocytes: pancreaticcarcinomas; neurons; endothelial cells; most astrocytomas keratinocytes;cells in possibly many other immunoprivileged tissues; cancers somecells in lungs, ovaries, liver, and prostate Myc-regulated Most lungcarcinomas Proliferating cells (only promoter (both small cell and somecell-types): non-small cell); most mammary epithelial cells colorectalcarcinomas (including non-proliferating) MAGE-1 Many melanomas; someTestis non-small cell lung carcinomas; some breast carcinomas VEGF 70%of all cancers Cells at sites of (constitutive neovascularizationoverexpression in (but unlike in tumors, many cancers) expression istransient, less strong, and never constitutive) bFGF Presumably manyCells at sites of ischemia different cancers, since (but unlike tumors,bFGF expression is expression is transient, induced by ischemic lessstrong, and never conditions constitutive) COX-2 Most colorectal Cellsat sites of carcinomas; many lung inflammation carcinomas; possibly manyother cancers IL-10 Most colorectal Leukocytes carcinomas; many lungcarcinomas; many squamous cell carcinomas of head and neck; possiblymany other cancers GRP78/BiP Presumably many Cells at sites of ishemiadifferent cancers, since GRP7S expression is induced by tumor- specificconditions CarG elements Induced by ionization Cells exposed to ionizingfrom Egr-1 radiation, so conceivably radiation; leukocytes most tumorsupon irradiation

2. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′□ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an TRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, herein incorporated by reference).

3. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites any of whichcan be used in conjunction with standard recombinant technology todigest the vector. (See Carbonelli et al., 1999, Levenson et al., 1998,and Cocea, 1997, incorporated herein by reference.) “Restriction enzymedigestion” refers to catalytic cleavage of a nucleic acid molecule withan enzyme that functions only at specific locations in a nucleic acidmolecule. Many of these restriction enzymes are commercially available.Use of such enzymes is widely understood by those of skill in the art.Frequently, a vector is linearized or fragmented using a restrictionenzyme that cuts within the MCS to enable exogenous sequences to beligated to the vector. “Ligation” refers to the process of formingphosphodiester bonds between two nucleic acid fragments, which may ormay not be contiguous with each other. Techniques involving restrictionenzymes and ligation reactions are well known to those of skill in theart of recombinant technology.

4. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the RNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In negative sense RNA viruses, including rhabdoviruses, termination isdefined by a RNA motif.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

5. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and/or any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal and/or the bovine growth hormone polyadenylationsignal, convenient and/or known to function well in various targetcells. Polyadenylation may increase the stability of the transcript ormay facilitate cytoplasmic transport.

6. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

7. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

D. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organisms that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors or viruses (which does notqualify as a vector if it expresses no exogenous polypeptides). A hostcell may be “transfected” or “transformed,” which refers to a process bywhich exogenous nucleic acid, such as a modified protein-encodingsequence, is transferred or introduced into the host cell. A transformedcell includes the primary subject cell and its progeny.

Host cells may be derived from prokaryotes or eukaryotes, includingyeast cells, insect cells, and mammalian cells, depending upon whetherthe desired result is replication of the vector or expression of part orall of the vector-encoded nucleic acid sequences. Numerous cell linesand cultures are available for use as a host cell, and they can beobtained through the American Type Culture Collection (ATCC), which isan organization that serves as an archive for living cultures andgenetic materials (www.atcc.org). An appropriate host can be determinedby one of skill in the art based on the vector backbone and the desiredresult. A plasmid or cosmid, for example, can be introduced into aprokaryote host cell for replication of many vectors. Bacterial cellsused as host cells for vector replication and/or expression includeDH5α, JM109, and KC8, as well as a number of commercially availablebacterial hosts such as SURE® Competent Cells and SOLOPACK™ Gold Cells(STRATAGENE®, La Jolla, Calif.). Alternatively, bacterial cells such asE. coli LE392 could be used as host cells for phage viruses. Appropriateyeast cells include Saccharomyces cerevisiae, Saccharomyces pombe, andPichia pastoris.

Examples of eukaryotic host cells for replication and/or expression of avector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Manyhost cells from various cell types and organisms are available and wouldbe known to one of skill in the art. Similarly, a viral vector may beused in conjunction with either a eukaryotic or prokaryotic host cell,particularly one that is permissive for replication or expression of thevector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

E. Expression Systems

Numerous expression systems exist that comprise at least all or part ofthe compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986 and 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH®.

In addition to the disclosed expression systems of the invention, otherexamples of expression systems include STRATAGENE®'s COMPLETE CONTROL™Inducible Mammalian Expression System, which involves a syntheticecdysone-inducible receptor, or its pET Expression System, an E. coliexpression system. Another example of an inducible expression system isavailable from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

F. Nucleic Acid Detection

In addition to their use in directing the expression of poxvirusproteins, polypeptides and/or peptides, the nucleic acid sequencesdisclosed herein have a variety of other uses. For example, they haveutility as probes or primers for embodiments involving nucleic acidhybridization. They may be used in diagnostic or screening methods ofthe present invention. Detection of nucleic acids encoding rhabdovirusor rhabdovirus polypeptide modulators are encompassed by the invention.

1. Hybridization

The use of a probe or primer of between 13 and 100 nucleotides,preferably between 17 and 100 nucleotides in length, or in some aspectsof the invention up to 1-2 kilobases or more in length, allows theformation of a duplex molecule that is both stable and selective.Molecules having complementary sequences over contiguous stretchesgreater than 20 bases in length are generally preferred, to increasestability and/or selectivity of the hybrid molecules obtained. One willgenerally prefer to design. nucleic acid molecules for hybridizationhaving one or more complementary sequences of 20 to 30 nucleotides, oreven longer where desired. Such fragments may be readily prepared, forexample, by directly synthesizing the fragment by chemical means or byintroducing selected sequences into recombinant vectors for recombinantproduction.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNAs and/or RNAs or to provide primers for amplification ofDNA or RNA from samples. Depending on the application envisioned, onewould desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of the probe or primers for the targetsequence,

For applications requiring high selectivity, one will typically desireto employ relatively high stringency conditions to form the hybrids. Forexample, relatively low salt and/or high temperature conditions, such asprovided by about 0.02 M to about 0.10 M NaCl at temperatures of about50° C. to about 70° C. Such high stringency conditions tolerate little,if any, mismatch between the probe or primers and the template or targetstrand and would be particularly suitable for isolating specific genesor for detecting specific mRNA transcripts. It is generally appreciatedthat conditions can be rendered more stringent by the addition ofincreasing amounts of formamide.

For certain applications, for example, site-directed mutagenesis, it isappreciated that lower stringency conditions are preferred. Under theseconditions, hybridization may occur even though the sequences of thehybridizing strands are not perfectly complementary, but are mismatchedat one or more positions. Conditions may be rendered less stringent byincreasing salt concentration and/or decreasing temperature. Forexample, a medium stringency condition could be provided by about 0.1 to0.25 M NaCl at temperatures of about 37° C. to about 55° C., while a lowstringency condition could be provided by about 0.15 M to about 0.9 Msalt, at temperatures ranging from about 20° C. to about 55° C.Hybridization conditions can be readily manipulated depending on thedesired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 1.0 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C.

In certain embodiments, it will be advantageous to employ nucleic acidsof defined sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of being detected. In preferredembodiments, one may desire to employ a fluorescent label or an enzymetag such as urease, alkaline phosphatase or peroxidase, instead ofradioactive or other environmentally undesirable reagents. In the caseof enzyme tags, colorimetric indicator substrates are known that can beemployed to provide a detection means that is visibly orspectrophotometrically detectable, to identify specific hybridizationwith complementary nucleic acid containing samples.

In general, it is envisioned that the probes or primers described hereinwill be useful as reagents in solution hybridization, as in PCR™, fordetection of expression of corresponding genes, as well as inembodiments employing a solid phase. In embodiments involving a solidphase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to hybridization with selected probes under desiredconditions. The conditions selected will depend on the particularcircumstances (depending, for example, on the G+C content, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Optimization of hybridization conditions for theparticular application of interest is well known to those of skill inthe art. After washing of the hybridized molecules to removenon-specifically bound probe molecules, hybridization is detected,and/or quantified, by determining the amount of bound label.Representative solid phase hybridization methods are disclosed in U.S.Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods ofhybridization that may be used in the practice of the present inventionare disclosed in U.S. Pat. Nos. 5,849,481, 5,849,486 and 5,851,772. Therelevant portions of these and other references identified in thissection of the Specification are incorporated herein by reference.

2. Amplification of Nucleic Acids

Nucleic acids used as a template for amplification may be isolated fromcells, tissues or other samples according to standard methodologies(Sambrook et al., 2001). In certain embodiments, analysis is performedon whole cell or tissue homogenates or biological fluid samples withoutsubstantial purification of the template nucleic acid. The nucleic acidmay be genomic DNA or fractionated or whole cell RNA. Where RNA is used,it may be desired to first convert the RNA to a complementary DNA.

The term “primer,” as used herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty and/or thirty base pairs in length, but longersequences can be employed. Primers may be provided in double-strandedand/or single-stranded form, although the single-stranded form ispreferred.

Pairs of primers designed to selectively hybridize to nucleic acidscorresponding to sequences of genes identified herein are contacted withthe template nucleic acid under conditions that permit selectivehybridization. Depending upon the desired application, high stringencyhybridization conditions may be selected that will only allowhybridization to sequences that are completely complementary to theprimers. In other embodiments, hybridization may occur under reducedstringency to allow for amplification of nucleic acids contain one ormore mismatches with the primer sequences. Once hybridized, thetemplate-primer complex is contacted with one or more enzymes thatfacilitate template-dependent nucleic acid synthesis. Multiple rounds ofamplification, also referred to as “cycles,” are conducted until asufficient amount of amplification product is produced.

A number of template dependent processes are available to amplify theoligonucleotide sequences present in a given template sample. One of thebest known amplification methods is the polymerase chain reaction(referred to as PCR™) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1988, each ofwhich is incorporated herein by reference in their entirety.

A reverse transcriptase PCR™ amplification procedure may be performed toquantify the amount of mRNA amplified and are well known (see Sambrooket al., 2001; WO 90/07641; and U.S. Pat. No. 5,882,864).

Another method for amplification is ligase chain reaction (“LCR”),disclosed in European Application No. 320 308, incorporated herein byreference in its entirety. U.S. Pat. No. 4,883,750 describes a methodsimilar to LCR for binding probe pairs to a target sequence. A methodbased on PCR™ and oligonucleotide ligase assay (OLA), disclosed in U.S.Pat. No. 5,912,148, may also be used. Alternative methods foramplification of target nucleic acid sequences that may be used in thepractice of the present invention are disclosed in U.S. Pat. Nos.5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497, 5,849,547,5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905, 5,928,906,5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB Application No. 2 202328, and in PCT Application No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety. Qbeta Replicase,described in PCT Application No. PCT/US87/00880, may also be used as anamplification method in the present invention. Isothermal amplificationas described by Walker et al. (1992) can also be used. As well as StrandDisplacement Amplification (SDA), disclosed in U.S. Pat. No. 5,916,779.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989; PCT Application WO88/10315, incorporated herein by reference in their entirety). EuropeanApplication No. 329 822 disclose a nucleic acid amplification processinvolving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA,and double-stranded DNA (dsDNA), which may be used in accordance withthe present invention.

PCT Application WO 89/06700 (incorporated herein by reference in itsentirety) disclose a nucleic acid sequence amplification scheme based onthe hybridization of a promoter region/primer sequence to a targetsingle-stranded DNA (“ssDNA”) followed by transcription of many RNAcopies of the sequence. Other amplification methods include “RACE” and“one-sided PCR” (Frohman, 1990; Ohara et al., 1989).

3. Detection of Nucleic Acids

Following any amplification, it may be desirable to separate and/orisolate the amplification product from the template and/or the excessprimer. In one embodiment, amplification products are separated byagarose, agarose-acrylamide, or polyacrylamide gel electrophoresis usingstandard methods (Sambrook et al., 2001).

Separation of nucleic acids may also be effected by chromatographictechniques known in art. There are many kinds of chromatography whichmay be used in the practice of the present invention, includingadsorption, partition, ion-exchange, hydroxylapatite, molecular sieve,reverse-phase, column, paper, thin-layer, and gas chromatography as wellas HPLC.

Typical visualization methods includes staining of a gel with ethidiumbromide and visualization of bands under UV light. Alternatively, if theamplification products are integrally labeled with radio- orfluorometrically-labeled nucleotides, the separated amplificationproducts can be exposed to x-ray film or visualized under theappropriate excitatory spectra.

In particular embodiments, detection is by Southern blotting andhybridization with a labeled probe. The techniques involved in Southernblotting are well known to those of skill in the art (see Sambrook etal., 2001). One example of the foregoing is described in U.S. Pat. No.5,279,721, incorporated by reference herein, which discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids.

Other methods of nucleic acid detection that may be used in the practiceof the instant invention are disclosed in U.S. Pat. Nos. 5,840,873,5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729,5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, 5,861,244,5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124,5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227,5,932,413 and 5,935,791, each of which is incorporated herein byreference.

4. Other Assays

Other methods for genetic screening may be used within the scope of thepresent invention, for example, to detect mutations in genomic nucleicacids, cDNA and/or RNA samples. Methods used to detect point mutationsinclude denaturing gradient gel electrophoresis (“DGGE”), restrictionfragment length polymorphism analysis (“RFLP”), chemical or enzymaticcleavage methods, direct sequencing of target regions amplified by PCR™(see above), single-strand conformation polymorphism analysis (“SSCP”)and other methods well known in the art. One method of screening forpoint mutations is based on RNase cleavage of base pair mismatches inRNA/DNA or RNA/RNA heteroduplexes. As used herein, the term “mismatch”is defined as a region of one or more unpaired or mispaired nucleotidesin a double-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. Thisdefinition thus includes mismatches due to insertion/deletion mutations,as well as single or multiple base point mutations (for example see U.S.Pat. No. 4,946,773. Alternative methods for detection of deletion,insertion or substitution mutations that may be used in the practice ofthe present invention are disclosed in U.S. Pat. Nos. 5,849,483,5,851,770, 5,866,337, 5,925,525 and 5,928,870, each of which isincorporated herein by reference in its entirety.

G. Methods of Gene Transfer

Suitable methods for nucleic acid delivery to effect expression ofcompositions of the present invention are believed to include virtuallyany method by which a nucleic acid (e.g., DNA or RNA, including viraland nonviral vectors) can be introduced into an organelle, a cell, atissue or an organism, as described herein or as would be known to oneof ordinary skill in the art. Such methods include, but are not limitedto, direct delivery of nucleic acid such as by injection (U.S. Pat. Nos.5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932,5,656,610, 5,589,466 and 5,580,859, each incorporated herein byreference), including microinjection (Harland and Weintraub, 1985; U.S.Pat. No. 5,789,215, incorporated herein by reference); byelectroporation (U.S. Pat. No. 5,384,253, incorporated herein byreference); by calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et at, 1990); by using DEAE dextranfollowed by polyethylene glycol (Gopal, 1985); by direct sonic loading(Fechheimer et al., 1987); by liposome mediated transfection (Nicolauand Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et at,1980; Kaneda et at, 1989; Kato et al., 1991); by microprojectilebombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat.Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880,and each incorporated herein by reference); by agitation with siliconcarbide fibers (Kaeppler et at, 1990; U.S. Pat. Nos. 5,302,523 and5,464,765, each incorporated herein by reference); by Agrobacteriummediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055, eachincorporated herein by reference); or by PEG mediated transformation ofprotoplasts (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and4,952,500, each incorporated herein by reference); bydesiccation/inhibition mediated DNA uptake (Potrykus et al., 1985).Through the application of techniques such as these, organelle(s),cell(s), tissue(s) or organism(s) may be stably or transientlytransformed.

H. Lipid Components and Moieties

In certain embodiments, the present invention concerns compositionscomprising one or more lipids associated with a nucleic acid, an aminoacid molecule, such as a peptide, or another small molecule compound. Inany of the embodiments discussed herein, the molecule may be either arhabdovirus polypeptide or a rhabdovirus polypeptide modulator, forexample a nucleic acid encoding all or part of either a rhabdoviruspolypeptide, or alternatively, an amino acid molecule encoding all orpart of rhabdovirus polypeptide modulator. A lipid is a substance thatis characteristically insoluble in water and extractable with an organicsolvent. Compounds other than those specifically described herein areunderstood by one of skill in the art as lipids, and are encompassed bythe compositions and methods of the present invention. A lipid componentand a non-lipid may be attached to one another, either covalently ornon-covalently.

A lipid may be naturally occurring or synthetic (i.e., designed orproduced by man). However, a lipid is usually a biological substance.Biological lipids are well known in the art, and include for example,neutral fats, phospholipids, phosphoglycerides, steroids, terpenes,lysolipids, glycosphingolipids, glucolipids, sulphatides, lipids withether and ester-linked fatty acids and polymerizable lipids, andcombinations thereof.

A nucleic acid molecule or amino acid molecule, such as a peptide,associated with a lipid may be dispersed in a solution containing alipid, dissolved with a lipid, emulsified with a lipid, mixed with alipid, combined with a lipid, covalently bonded to a lipid, contained asa suspension in a lipid or otherwise associated with a lipid. A lipid orlipid/virus-associated composition of the present invention is notlimited to any particular structure. For example, they may also simplybe interspersed in a solution, possibly forming aggregates which are notuniform in either size or shape. In another example, they may be presentin a bilayer structure, as micelles, or with a “collapsed” structure. Inanother non-limiting example, a lipofectamine (Gibco BRL)-poxvirus orSuperfect (Qiagen)-virus complex is also contemplated.

In certain embodiments, a lipid composition may comprise about 1%, about2%, about 3%, about 4% about 5%, about 6%, about 7%, about 8%, about 9%,about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%,about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%,about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%,about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%,about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%,or any range derivable therein, of a particular lipid, lipid type, ornon-lipid component such as a chug, protein, sugar, nucleic acids orother material disclosed herein or as would be known to one of skill inthe art. In a non-limiting example, a lipid composition may compriseabout 10% to about 20% neutral lipids, and about 33% to about 34% of acerebroside, and about 1% cholesterol. Thus, it is contemplated thatlipid compositions of the present invention may comprise any of thelipids, lipid types, or other components in any combination orpercentage range.

IV. PHARMACEUTICAL FORMULATIONS AND TREATMENT REGIMENS

In an embodiment of the present invention, a method of treatment for ahyperproliferative or neoplastic disease, such as cancer, by thedelivery of a non-VSV rhabdovirus, such as Maraba virus, Carajas virus,Muir Springs virus, and/or Bahia Grande virus, is contemplated. Examplesof cancer contemplated for treatment include lung cancer, head and neckcancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer,bone cancer, testicular cancer, cervical cancer, gastrointestinalcancer, lymphomas, pre-neoplastic lesions, pre-neoplastic lesions in thelung, colon cancer, melanoma, bladder cancer and any other cancers ortumors that may be treated, including metastatic or systemicallydistributed cancers.

An effective amount of the pharmaceutical composition, generally, isdefined as that amount sufficient to detectably and repeatedly to slow,ameliorate, reduce, minimize, or limit. the extent of the disease or itssymptoms. More rigorous definitions may apply, including elimination,eradication, or cure of disease.

Preferably, patients will have adequate bone marrow function (defined asa peripheral absolute granulocyte count of >2,000/mm³ and a plateletcount of 100,000/mm³), adequate liver function (bilirubin <1.5 mg/dl)and adequate renal function (creatinine <1.5 mg/dl).

A. Administration

To kill cells, inhibit cell growth, inhibit metastasis, decrease tumoror tissue size, and otherwise reverse, stay, or reduce the malignantphenotype of tumor cells, using the methods and compositions of thepresent invention, one would generally contact a hyperproliferative orneoplastic cell with a therapeutic composition such as a virus or anexpression construct encoding a polypeptide. The routes ofadministration will vary, naturally, with the location and nature of thelesion, and include, e.g., intradermal, transdermal, parenteral,intravascular, intravenous, intramuscular, intranasal, subcutaneous,regional, percutaneous, intratracheal, intraperitoneal, intraarterial,intravesical, intratumoral, inhalation, perfusion, lavage, directinjection, alimentary, and oral administration and formulation.

To effect a therapeutic benefit with respect to a vascular condition ordisease, one would contact a vascular cell with the therapeuticcompound. Any of the formulations and routes of administration discussedwith respect to the treatment or diagnosis of cancer may also beemployed with respect to vascular diseases and conditions.

Intratumoral injection, or injection into the tumor vasculature iscontemplated for discrete, solid, accessible tumors. Local, regional orsystemic administration is also contemplated, particularly for thosecancers that are disseminated or are likely to disseminatedsystemically. The viral particles may be administering by at least or atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 injections.

In the case of surgical intervention, the present invention may be usedpreoperatively, to render an inoperable tumor subject to resection.Alternatively, the present invention may be used at the time of surgery,and/or thereafter, to treat residual or metastatic disease. For example,a resected tumor bed may be injected or perfused with a formulationcomprising a rhabdovirus polypeptide or a rhabdovirus, which may or maynot harbor a mutation, that is advantageous for treatment of cancer orcancer cells. The perfusion may be continued post-resection, forexample, by leaving a catheter implanted at the site of the surgery.Periodic post-surgical treatment also is envisioned.

Continuous administration also may be applied where appropriate, forexample, where a tumor is excised and the tumor bed is treated toeliminate residual, microscopic disease. Delivery via syringe orcatherization is preferred. Such continuous perfusion may take place fora period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours,to about 12-24 hours, to about 1-2 days, to about 1-2 wk or longerfollowing the initiation of treatment. Generally, the dose of thetherapeutic composition via continuous perfusion will be equivalent tothat given by a single or multiple injections, adjusted over a period oftime during which the perfusion occurs. It is further contemplated thatlimb perfusion may be used to administer therapeutic compositions of thepresent invention, particularly in the treatment of melanomas andsarcomas.

Treatment regimens may vary as well, and often depend on tumor type,tumor location, disease progression, and health and age of the patient.Obviously, certain types of tumor will require more aggressivetreatment, while at the same time, certain patients cannot tolerate moretaxing protocols. The clinician will be best suited to make suchdecisions based on the known efficacy and toxicity (if any) of thetherapeutic formulations.

In certain embodiments, the tumor being treated may not, at leastinitially, be resectable. Treatments with therapeutic viral constructsmay increase the resectability of the tumor due to shrinkage at themargins or by elimination of certain particularly invasive portions.Following treatments, resection may be possible. Additional treatmentssubsequent to resection will serve to eliminate microscopic residualdisease at the tumor site.

A typical course of treatment, for a primary tumor or a post-excisiontumor bed, will involve multiple doses. Typical primary tumor treatmentinvolves a 1, 2, 3, 4, 5, 6 or more dose application over a 1, 2, 3, 4,5, 6-week period or more. A two-week regimen may be repeated one, two,three, four, five, six or more times. During a course of treatment, theneed to complete the planned dosings may be re-evaluated.

The treatments may include various “unit doses.” Unit dose is defined ascontaining a predetermined quantity of the therapeutic composition. Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. A unit dose need notbe administered as a single injection but may comprise continuousinfusion over a set period of time. Unit dose of the present inventionmay conveniently be described in terms of plaque forming units (pfu) orviral particles for viral constructs. Unit doses range from 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ pfu or vp and higher.Alternatively, depending on the kind of virus and the titer attainable,one will deliver 1 to 100, 10 to 50, 100-1000, or up to about 1×10⁴,1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³,1×10¹⁴, or 1×10¹⁵ or higher infectious viral particles (vp) to thepatient or to the patient's cells.

B. Injectable Compositions and Formulations

The preferred method for the delivery of an expression construct orvirus encoding all or part of a rhabdovirus genome to cancer or tumorcells in the present invention is via intravascular injection. However,the pharmaceutical compositions disclosed herein may alternatively beadministered intratumorally, parenterally, intravenously,intrarterially, intradermally, intramuscularly, transdermally or evenintraperitoneally as described in U.S. Pat. Nos. 5,543,158, 5,641,515and 5,399,363 (each specifically incorporated herein by reference in itsentirety).

Injection of nucleic acid constructs may be delivered by syringe or anyother method used for injection of a solution, as long as the expressionconstruct can pass through the particular gauge of needle required forinjection (for examples see U.S. Pat. Nos. 5,846,233 and 5,846,225).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, intratumoral, and intraperitonealadministration. In this connection, sterile aqueous media that can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage may be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Moreover, for human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsrequired by governments of the countries in which the compositions arebeing used.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug release capsules and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” or“pharmacologically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared.

C. Combination Treatments

The compounds and methods of the present invention may be used in thecontext of hyperproliferative or neoplastic diseases/conditionsincluding cancer and atherosclerosis. In order to increase theeffectiveness of a treatment with the compositions of the presentinvention, such as rhabdoviruses, it may be desirable to combine thesecompositions with other agents effective in the treatment of thosediseases and conditions. For example, the treatment of a cancer may beimplemented with therapeutic compounds of the present invention andother anti-cancer therapies, such as anti-cancer agents or surgery.

Various combinations may be employed; for example, a non-VSVrhabdovirus, such as Maraba virus, Carajas virus, Muir Springs virus,and/or Bahia Grande virus, is “A” and the secondary anti-cancer therapyis “B”, which may include a second rhabdovirus:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of the therapeutic virus or viral constructs of thepresent invention to a patient will follow general protocols for theadministration of that particular secondary therapy, taking into accountthe toxicity, if any, of the virus treatment. It is expected that thetreatment cycles would be repeated as necessary. It also is contemplatedthat various standard therapies, as well as surgical intervention, maybe applied in combination with the described cancer or tumor celltherapy.

1. Anti-Cancer Therapy

An “anti-cancer” agent is capable of negatively affecting cancer in asubject, for example, by killing cancer cells, inducing apoptosis incancer cells, reducing the growth rate of cancer cells, reducing theincidence or number of metastases, reducing tumor size, inhibiting tumorgrowth, reducing the blood supply to a tumor or cancer cells, promotingan immune response against cancer cells or a tumor, preventing orinhibiting the progression of cancer, or increasing the lifespan of asubject with cancer. Anti-cancer agents include biological agents(biotherapy), chemotherapy agents, and radiotherapy agents. Moregenerally, these other compositions would be provided in a combinedamount effective to kill or inhibit proliferation of the cell. Thisprocess may involve contacting the cells with virus or viral constructand the agent(s) or multiple factor(s) at the same time. This may beachieved by contacting the cell with a single composition orpharmacological formulation that includes both agents, or by contactingthe cell with two distinct compositions or formulations, at the sametime, wherein one composition includes the virus and the other includesthe second agent(s).

Tumor cell resistance to chemotherapy and radiotherapy agents representsa major problem in clinical oncology. One goal of current cancerresearch is to find ways to improve the efficacy of chemo- andradiotherapy by combining it with gene therapy. For example, the herpessimplex-thymidine kinase (HS-tK) gene, when delivered to brain tumors bya retroviral vector system, successfully induced susceptibility to theantiviral agent ganciclovir (Culver et al., 1992). In the context of thepresent invention, it is contemplated that poxvirus therapy could beused similarly in conjunction with chemotherapeutic, radiotherapeutic,immunotherapeutic, or other biological intervention, in addition toother pro-apoptotic or cell cycle regulating agents.

Alternatively, a viral therapy may precede or follow the other treatmentby intervals ranging from minutes to weeks. In embodiments where theother agent and virus are applied separately to the cell, one wouldgenerally ensure that a significant period of time did not expirebetween the time of each delivery, such that the agent and virus wouldstill be able to exert an advantageously combined effect on the cell. Insuch instances, it is contemplated that one may contact the cell withboth modalities within about 12-24 h of each other and, more preferably,within about 6-12 h of each other. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

a. Chemotherapy

Cancer therapies also include a variety of combination therapies withboth chemical and radiation based treatments. Combination chemotherapiesinclude, for example, cisplatin (CDDP), carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),tamoxifen, raloxifene, estrogen receptor binding agents, taxol,gemcitabien, navelbine, farnesyl-protein transferase inhibitors,transplatinum, 5-fluorouracil, vincristine, vinblastine andmethotrexate, Temazolomide (an aqueous form of DTIC), or any analog orderivative variant of the foregoing. The combination of chemotherapywith biological therapy is known as biochemotherapy.

b. Radiotherapy

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, proton beams, and/orthe directed delivery of radioisotopes to tumor cells. Other forms ofDNA damaging factors are also contemplated such as microwaves andUV-irradiation. It is most likely that all of these factors effect abroad range of damage on DNA, on the precursors of DNA, on thereplication and repair of DNA, and on the assembly and maintenance ofchromosomes. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (3 to 4 wk), to single dosesof 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely,and depend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by the neoplastic cells.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

c. Immunotherapy

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells. The combination of therapeuticmodalities, i.e., direct cytotoxic activity and inhibition or reductionof certain rhabdovirus or rhabdovirus polypeptides would providetherapeutic benefit in the treatment of cancer.

Immunotherapy could also be used as part of a combined therapy. Thegeneral approach for combined therapy is discussed below. In one aspectof immunotherapy, the tumor cell must bear some marker that is amenableto targeting, i.e., is not present on the majority of other cells. Manytumor markers exist and any of these may be suitable for targeting inthe context of the present invention. Common tumor markers includecarcinoembryonic antigen, prostate specific antigen, urinary tumorassociated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG,Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B and p155. Tumor cell lysates may also be used in anantigenic composition.

An alternative aspect of immunotherapy is to combine anticancer effectswith immune stimulatory effects. Immune stimulating molecules include:cytokines such as IL-2, IL-4, IL-12, GM-CSF, IFNγ, chemokines such asMIP-1, MCP-1, IL-8 and growth factors such as FLT3 ligand. Combiningimmune stimulating molecules, either as proteins or using gene deliveryin combination with a tumor suppressor has been shown to enhanceanti-tumor effects (Ju et al., 2000).

As discussed earlier, examples of immunotherapies currently underinvestigation or in use are immune adjuvants (e.g., Mycobacterium bovis,Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds)(U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998;Christodoulides et al., 1998), cytokine therapy (e.g., interferons α, βand γ; IL-1, GM-CSF and TNF) (Bukowski et al., 1998; Davidson et al.,1998; Hellstrand et al., 1998) gene therapy (e.g., TNF, IL-1, IL-2, p53)(Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. Nos.5,830,880 and 5,846,945) and monoclonal antibodies (e.g.,anti-ganglioside GM2, anti-HER-2, anti-p185) (Pietras et al., 1998;Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311). Herceptin(trastuzumab) is a chimeric (mouse-human) monoclonal antibody thatblocks the HER2-neu receptor (Dillman, 1999). Combination therapy ofcancer with herceptin and chemotherapy has been shown to be moreeffective than the individual therapies. Thus, it is contemplated thatone or more anti-cancer therapies may be employed with therhabdovirus-related therapies described herein.

(1) Passive Immunotherapy

A number of different approaches for passive immunotherapy of cancerexist. They may be broadly categorized into the following: injection ofantibodies alone; injection of antibodies coupled to toxins orchemotherapeutic agents; injection of antibodies coupled to radioactiveisotopes; injection of anti-idiotype antibodies; and finally, purging oftumor cells in bone marrow.

Preferably, human monoclonal antibodies are employed in passiveimmunotherapy, as they produce few or no side effects in the patient.However, their application is somewhat limited by their scarcity andhave so far only been administered intralesionally. Human monoclonalantibodies to ganglioside antigens have been administeredintralesionally to patients suffering from cutaneous recurrent melanoma(Irie and Morton, 1986). Regression was observed in six out of tenpatients, following, daily or weekly, intralesional injections. Inanother study, moderate success was achieved from intralesionalinjections of two human monoclonal antibodies (Irie et al., 1989).

It may be favorable to administer more than one monoclonal antibodydirected against two different antigens or even antibodies with multipleantigen specificity. Treatment protocols also may include administrationof lymphokines or other immune enhancers as described by Bajorin et al.(1988). The development of human monoclonal antibodies is described infurther detail elsewhere in the specification.

(2) Active Immunotherapy

In active immunotherapy, an antigenic peptide, polypeptide or protein,or an autologous or allogenic tumor cell composition or “vaccine” isadministered, generally with a distinct bacterial adjuvant (Ravindranathand Morton, 1991; Morton et al., 1992; Mitchell et al., 1990; Mitchellet al., 1993). In melanoma immunotherapy, those patients who elicit highIgM response often survive better than those who elicit no or low IgMantibodies (Morton et al., 1992). IgM antibodies are often transientantibodies and the exception to the rule appears to be anti gangliosideor anticarbohydrate antibodies.

(³) Adoptive Immunotherapy

In adoptive immunotherapy, the patient's circulating lymphocytes, ortumor infiltrated lymphocytes, are isolated in vitro, activated bylymphokines such as IL 2 or transduced with genes for tumor necrosis,and readministered (Rosenberg et al., 1988; 1989). To achieve this, onewould administer to an animal, or human patient, an immunologicallyeffective amount of activated lymphocytes in combination with anadjuvant incorporated antigenic peptide composition as described herein.The activated lymphocytes will most preferably be the patient's owncells that were earlier isolated from a blood or tumor sample andactivated (or “expanded”) in vitro. This form of immunotherapy hasproduced several cases of regression of melanoma and renal carcinoma,but the percentage of responders were few compared to those who did notrespond.

d. Genes

In yet another embodiment, the secondary treatment is a gene therapy inwhich a therapeutic polynucleotide is administered before, after, or atthe same time as a rhabdovirus is administered. Delivery of arhabdovirus in conjunction with a vector encoding one of the followinggene products will have a combined anti-cancer effect on target tissues.Alternatively, the rhabdovirus may be engineered as a viral vector toinclude the therapeutic polynucleotide. A variety of proteins areencompassed within the invention, some of which are described below.Table 4 lists various genes that may be targeted for gene therapy ofsome form in combination with the present invention.

(1) Inducers of Cellular Proliferation

The proteins that induce cellular proliferation further fall intovarious categories dependent on function. The commonality of all ofthese proteins is their ability to regulate cellular proliferation. Forexample, a form of PDGF, the sis oncogene, is a secreted growth factor.Oncogenes rarely arise from genes encoding growth factors, and at thepresent, sis is the only known naturally-occurring oncogenic growthfactor. In one embodiment of the present invention, it is contemplatedthat anti-sense mRNA directed to a particular inducer of cellularproliferation is used to prevent expression of the inducer of cellularproliferation.

(2) Inhibitors of Cellular Proliferation

The tumor suppressor oncogenes function to inhibit excessive cellularproliferation. The inactivation of these genes destroys their inhibitoryactivity, resulting in unregulated proliferation. Tumor suppressorsinclude p53, p16 and C-CAM. Other genes that may be employed accordingto the present invention include Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I,MEN-II, zac1, p73, VHL, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16fusions, p21/p27 fusions, anti-thrombotic genes (e.g., COX-1, TFPI),PGS, Dp, E2F, ras, myc, ncu, raf, erb, fms, trk, ret, gsp, hst, abl,E1A, p300, genes involved in angiogenesis (e.g., VEGF, FGF,thrombospondin, BAI-1, GDAIF, or their receptors) and MCC.

(3) Regulators of Programmed Cell Death

Apoptosis, or programmed cell death, is an essential process for normalembryonic development, maintaining homeostasis in adult tissues, andsuppressing carcinogenesis (Kerr et al., 1972). The Bcl-2 family ofproteins and ICE-like proteases have been demonstrated to be importantregulators and effectors of apoptosis in other systems. The Bcl 2protein, discovered in association with follicular lymphoma, plays aprominent role in controlling apoptosis and enhancing cell survival inresponse to diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary andSklar, 1985; Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto andCroce, 1986). The evolutionarily conserved Bcl-2 protein now isrecognized to be a member of a family of related proteins, which can becategorized as death agonists or death antagonists.

Subsequent to its discovery, it was shown that Bcl 2 acts to suppresscell death triggered by a variety of stimuli. Also, it now is apparentthat there is a family of Bcl-2 cell death regulatory proteins whichshare in common structural and sequence homologies. These differentfamily members have been shown to either possess similar functions toBcl 2 (e.g., BclXL, BclW, BclS, Mcl-1, A1, Bfl-1) or counteract Bcl 2function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad,Harakiri).

c. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and microscopically controlled surgery(Mohs' surgery). It is further contemplated that the present inventionmay be used in conjunction with removal of superficial cancers,pre-cancers, or incidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

f. Other Agents

It is contemplated that other agents may be used in combination with thepresent invention to improve the therapeutic efficacy of treatment.These additional agents include immunomodulatory agents, agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion,agents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers, or other biological agents. Immunomodulatory agentsinclude tumor necrosis factor; interferon α, β, and γ; IL-2 and othercytokines; F42K and other cytokine analogs; or MIP-1, MIP-1β, MCP-1,RANTES, and other chemokines. It is further contemplated that theupregulation of cell surface receptors or their ligands such as Fas/Fasligand, DR4 or DR5/TRAIL (Apo-2 ligand) would potentiate the apoptoticinducing ability of the present invention by establishment of anautocrine or paracrine effect on hyperproliferative cells. Increasesintercellular signaling by elevating the number of GAP junctions wouldincrease the anti-hyperproliferative effects on the neighboringhyperproliferative cell population. In other embodiments, cytostatic ordifferentiation agents can be used in combination with the presentinvention to improve the anti-hyperproliferative efficacy of thetreatments. Inhibitors of cell adhesion are contemplated to improve theefficacy of the present invention. Examples of cell adhesion inhibitorsare focal adhesion kinase (FAKs) inhibitors and Lovastatin. It isfurther contemplated that other agents that increase the sensitivity ofa hyperproliferative cell to apoptosis, such as the antibody c225, couldbe used in combination with the present invention to improve thetreatment efficacy.

There have been many advances in the therapy of cancer following theintroduction of cytotoxic chemotherapeutic drugs. However, one of theconsequences of chemotherapy is the development/acquisition ofdrug-resistant phenotypes and the development of multiple drugresistance. The development of drug resistance remains a major obstaclein the treatment of such tumors and therefore, there is an obvious needfor alternative approaches such as viral therapy.

Another form of therapy for use in conjunction with chemotherapy,radiation therapy or biological therapy includes hyperthermia, which isa procedure in which a patient's tissue is exposed to high temperatures(up to 106° F.). External or internal heating devices may be involved inthe application of local, regional, or whole-body hyperthermia. Localhyperthermia involves the application of heat to a small area, such as atumor. Heat may be generated externally with high-frequency wavestargeting a tumor from a device outside the body. Internal heat mayinvolve a sterile probe, including thin, heated wires or hollow tubesfilled with warm water, implanted microwave antennae, or radiofrequencyelectrodes.

A patient's organ or a limb is heated for regional therapy, which isaccomplished using devices that produce high energy, such as magnets.Alternatively, some of the patient's blood may be removed and heatedbefore being perfused into an area that will be internally heated.Whole-body heating may also be implemented in cases where cancer hasspread throughout the body. Warm-water blankets, hot wax, inductivecoils, and thermal chambers may be used for this purpose.

Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment

V. EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. The present examples, along with themethods described herein are presently representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

Example 1 Screening for Novel Oncolytic Candidate Rhabdoviruses

In vitro screens. As an initial screen to identify novel oncolyticviruses, rhabdovirus field isolates were assessed for their ability tokill human tumor cells from the NCI 60 cell panel. This has been afruitful strategy ⁻for the inventors in the past to determine therelative effectiveness of a series of VSV mutants as oncolytic (cancercell lysing) candidates. Initially, the inventors have examined 13 novelrhabdoviruses that have been previously determined to replicate inmammalian cells. It is contemplated that this procedure will be extendedto study rhabdoviruses for which there is less experience in cellculture. In an effort to rapidly and efficiently screen through a matrixof 60 cells infected with 13 different viruses, the inventors use arapid and inexpensive assay in 96 well format using MTS reduction toformazan, or crystal violet staining of residual cells, to measure cellnumber and viability. The inventors grow cell lines to 80% confluence in96 well plates and then expose them in parallel to our rhabdovirus fieldisolates at increasing MOIs (MOI=0.0001-10 PFUs/cell). At 48 and 96hours post infection, cells are stained with aqueous MTS regent (PromegaUSA) and incubated for 3 hours to allow sufficient formazan formation.Alternatively, the plates of infected cells are washed with buffer toremove dead cells, stained with crystal violet dye, washed to removeresidual dye, after which time the dye is solublized using detergent.These plates are then read using the integrated multiwell plate reader(Biotek SynergyHT; USA), the data curve fitted, and the EC₅₀ determinedfrom this curve. Typically, assays are performed in sextuplet, with thehighest and lowest EC₅₀ values removed, and averaging the remaining fourEC₅₀ to ultimately determine a value and confidence interval. (Forexample see FIG. 2)

As a counter screen to assess whether a particular virus infects/killsnormal human cells in vitro, cultures of normal human fibroblasts,epithelium and endothelium and neuronal cultures from the inventorscollection and those commercially available (Cambrex, USA) will bescreened. Cultures will be infected with candidate viruses (0.1 to 20pfu/cell) for 48 and 96 hours. Cell viability will be detected by MTSassay, or crystal violet assay, and further characterized by labelingwith activated caspase 3 antibody D175 (Cell Signaling Technologies,USA) and detected using a FITC-conjugated secondary antibody. Studieswill be done in parallel with known susceptible/resistant human andmouse tumor cell lines. A combination of untreated cells and cellstreated with TRAIL and cyclohexamide has been used to establish thedynamic range of the assay, with preliminary z-factor determinationssignificantly above 0.5.

Another contingency is that viruses may replicate and spread efficientlywithin cultures without rapidly killing these cells. These are alsopotentially interesting viruses, provided their replication is tumorselective in nature, as their lytic capacity could subsequently beincreased through recombinant engineering. To detect these viruses, theinventors will infect cells of the NCI 60 cell panel with field isolatesat a low MOI (0.1 pfu/cell) in duplicate wells of a 24 well plate. After1 hour, wells will be washed thoroughly to remove free input virus,medium added and the cultures incubated for a further 72 hours. Theseculture supernatants will subsequently be titered on a permissive cellline (Vero cells) to detect and quantify productive infection. The finalwash from each of these will be titered to control for residual inputvirus. Candidate virus hits in this assay will be confirmed in tissueculture cells using virus-specific antisera and standardimmunofluorescence microscopy.

Rank based on all parameters. Several properties contribute to oncolytickilling of tumor cells including: ability to induce apoptosis, rate ofvirus production, quantity of virus produced, as well as specialfunctions such as syncytia formation. Promising candidates from theinitial screen will be characterized further with respect to apoptosisinduction (as determined by TUNEL assay and immunofluorescence stainingfor activated caspase-3), and one step growth curves to compare kineticsand to quantify virus production. These studies will serve as a guide toimproving these strains. For example: (1) if a virus kills tumor cellswell but shows unacceptable toxicity to normal cells, the inventors willattenuate this virus using one or more of the strategies outline below;(2) alternatively, if a virus shows slower killing kinetics whilemaintaining a high replication rate, then the inventors may add a toxicor therapeutic transgene; (3) If a candidate virus replicates slowly yetis an effective killer, the inventor will select a variant withincreased growth kinetics to boost its potency.

From the inventors experience with VSV and other oncolytic viruses, theyhave identified three key in vitro gating criteria to narrow the list ofcandidates: (1) selective tumor cell killing, (2) productive replicationwithin tumor cells (independent of killing), and (3) efficacy on VSVresistant tumor lines (UACC-62 melanoma, A431 and NCI-H226 lung, DU-145prostate, HL60 leukemia). Based on these criteria, results from thescreening assays described above will be integrated to pare the list forfurther evaluate in preliminary in vivo testing.

In vivo Toxicity and Biodistribution. The two routes of administrationrelated to a clinical setting are intravenous (IV) and intracranial (IC)injections. Lead candidates identified during in vitro screening fortoxicity and biodistribution in mice following infection will beassessed by these routes. Groups of 3 mice will be infected either by IVat doses of 1×10⁵ to 1×10⁹ pfu, or by IC at 1×10² to 1×10⁶ pfu. Inaddition to mortality, morbidity will be monitored daily for signs oflethargy, dehydration, weight loss and limb paralysis. Histopathologywill be performed on 2 mice from the minimum lethal dose group (highestdose if no lethal dose is achieved) from each candidate virus infection.WT VSV and mock infection will serve as appropriate positive andnegative controls respectively. Organs will be harvested from theremaining mouse in this group, homogenized and titered as a preliminaryassessment of virus biodistribution.

For viruses that display an acceptable lethal dose range, the inventorswill subsequently assess biodistribution in tumor bearing mice toidentify viruses compatible with systemic administration. The inventorwill employ three of our existing cancer models representing verydifferent organ targets of critical clinical relevance: (1) CT-26 mousecolon carcinoma (1×10⁵ cells) injected intravenously to formdisseminated lungs tumors in syngeneic Balb/C mice (2), 4T1 mouse breastcarcinoma (4×10⁵ cells) injected into the fat pad of syngeneic Balb/Cmice to form a single primary tumor with spontaneous metastases, and (3)U87 human glioblastoma cells (1×10⁵ cells) stereotactically implanted inthe cortex of nude mice. A maximum tolerable dose for each virus androute (IV or IC) will be determined from the preliminary in vivotoxicity experiments. This value will serve as an initial therapeuticdose for biodistribution studies in tumor bearing mice. In groups of 3mice, tumors will be established for 1 week and then treated IV or ICwith a single dose of each candidate virus at their respective MTD.Forty-eight hours post treatment, animals will be perfused with salineto flush any free virus from the circulation, and tumors and organs willbe harvested, homogenized and titered to quantify infectious virus. Inthis fashion, the inventors will determine which viruses can bedelivered to tumor sites by systemic injection, as well as the relativetumor selectivity of virus replication in vivo.

Re-Rank. Based on the toxicity, biodistribution, systemic delivery andtumor selectivity profiles in in vivo studies, the inventors will selectthe best candidates to proceed with detailed characterization andfurther development.

Example 2 Building Recombinants

Sequencing and Recombinant System. In order to facilitate rapid researchand development, subsequent production of clinical material and toensure the safety and stability of therapeutic viruses, the inventorswill clone and rescue recombinant forms selected viruses.

Many negative strand ssRNA viruses have been cloned and rescued usingstandard recombinant techniques. The inventors will employ similarstrategies that have been adopted successfully for reported recombinant−ssRNA viruses. Briefly, the genome of a candidate virus will beisolated by RNA extraction (Qiagen Corp) from 1×10⁹ virus purifiedparticles. The purified genomic RNA is then primed with random hexamersand reverse transcribed to cDNA, subsequently rendered double-strandedand cloned by ligating EcoRI adapters, size fractionated and finallyligating into an EcoRI digested bacterial plasmid (pT7Blue; Novagen).The result is a library of genomic fragments that can be easilysequenced by standard techniques. Because of the random primed nature ofthis library, this strategy will not “capture” the extreme 3′ and 5′ends. To do this the inventors ligate oligos to the 3′ or 5′ ends of thepurified genomic RNA using T4 RNA ligase. Using primers complementary tothe newly ligated oligo flanking the genome, the inventors PCR amplifyand clone the ends of the genome for subsequent sequencing. Thissequence information is then used to design end-specific primers foramplifying the entire genome, which is then cloned into a specializedplasmid. This plasmid flanks the genome with a T7 promoter on one endand a hepatitis delta self-cleaving ribozyme and T7 terminator sequenceon the opposite flank. When transfected into T7 RNA polymeraseexpressing (previously infected with a T7 expressing vaccinia virus)A549 cells, this plasmid generates viral genomes in the cytoplasm. Inparallel, the viruses' coding sequences for N, P and L genes are clonedinto CMV promoter driven expression plasmids. Co-transfection of thegenome construct with the N, P and L plasmids into these A549 cellsreconstitutes the viral replication complex on the viral genome andresults in rescue of infectious virus. As a proof of principle theinventors have cloned, genetically manipulated, and rescued Maraba virususing this method. See FIG. 17 and FIG. 18 for examples of Marabarelated viruses.

Example 3 Optimization/Augmentation

The non-VSV rhabdoviruses are feral viruses; and as with all oncolyticviruses reported thus far, including VSV, the inventors predict thatthese field isolates will benefit from further optimization through invitro selection and/or recombinant engineering strategies. Somecandidates may require attenuation (e.g., Maraba virus) while some mayrequire augmentation of their replication and/or tumor killing kinetics(e.g., Muir Springs virus). The following is a summary of severalstrategies the inventors will employ to maximize the effectiveness ofnewly identified therapeutic viruses.

Engineered Mutations. VSV blocks nuclear/cytoplasmic mRNA transport as ameans to defeat host cell innate immunity. The inventors have previouslydescribed engineering mutations into the M protein of VSV to disablethis activity and thereby selectively attenuate this virus in normalcells. Given that other members of the vesiculoviruses genus have alsodemonstrated this ability (Chandipura, and spring viremia of carp) andthat most vesiculoviruses sequenced thus far (VSV, Chandripura, Piry,Cocal, spring viremia of carp, Maraba) have the critical sequence motifrequired by VSV for this function, the inventors contemplate attenuateof non-VSV rhabdovirus in an analogous fashion to that used for VSV.However, other rhabdoviruses such as rabies and bovine ephemeral fevervirus do not have this motif and do not block nuclear cytoplasmic mRNAtransport and perhaps will not be amenable to this strategy ofattenuation. As more information becomes available regardingrhabdovirus/host interaction from consortium labs and others, additionalstructure/functioned-guided manipulations to attenuate theses viruseswill be possible.

Transgenes. There are now several reports of “arming” oncolytic viruseswith suicide genes or immune mediators to increase their potency. Theinventors will focus on adding transgenes to increase the cytotoxicityof candidate viruses that show efficient replication, but insufficienttumor killing. The inventors have a priority-weighted list of transgenesthat are currently being engineered into Maraba virus. At present theranking consists of: (1) Apoptosis Inducing Factor (AIF)—anoxido-reductase homolog responsible for chromatin collapse anddegradation in a caspase-independent manner. (2) HaraKiri—the mostpotent of the BH3-only pro-apoptotic member of the Bcl-2 familyresponsible for induction of conventional caspase-dependent apoptosis(Type I PCD). (3) XAF1—a potent tumor suppressor gene and directinhibitor of the IAP family. (4) Atg4B—the key protease responsible forinitiating autophagy (Type II PCD).

Ultimately, members of the intrinsic or extrinsic pathways of cell deathcould be engineered with Tat or other protein transduction domains to besecreted from virus infected cells to induce bystander killing withinthe tumor mass. The inventors remain cognizant that other bystanderkilling effects maybe mediated through components of the host immunityto virus and/or tumor. Thus an alternative strategy would be to engineera transgene(s) to draw immune cells to sites of infection. Evidenceindicates that virus infection of CT26 lung tumors induces neutrophilsto infiltrate the tumor and cause a massive apoptotic bystander killingeffect.

Directed evolution to improve oncolytic Rhabdoviruses. Many examples ofdirected evolution have been described where the replication fitness ofa parental virus strain was either increased or decreased by serialpassage in mammalian cell culture. Rhabdoviruses are particularlyamenable to this type of procedure as they exist not as a single entity,but as a population of strains called a quasi-species. The members ofthe quasi-species represent point mutants of the dominant genome. Whenan appropriate selection pressure is applied, the fittest member of thepopulation is selected for, and becomes the dominant genome. This hastremendous utility in efforts to build a better oncolytic virus becauseit provides one with a ready-made collection of mutants from which toselect a variant with better oncolytic capabilities. Thus, to attenuatea given candidate, the inventors will select small plaque mutants onprimary fibroblasts and subsequently amplify this cloned virus on tumorcells to back-select against non-productive mutations (i.e., mutationswhich uniformly debilitate, such as polymerase mutations, as opposed tospecific disabilities in normal cells/tissues). By performing this initerative cycles at high MOI (10 pfu/cell), the inventors expect toisolate a mutant that maintains robust replication in tumor cells, yethas lost the ability to productively infect healthy normal cells.Alternatively, the inventors may augment the potency of non-VSVrhabdoviruses, either by selecting faster replicators, or more lethalkillers. To speed up the replication rate of a candidate virus theinventors will perform iterative rounds of infection/replication intumor cell lines, but at each subsequent round will decrease the postinfection harvest time. This selection pressure will force viruses toevolve towards rapid replication. If enhanced cytotoxicity is desirable,the inventors will infect resistant or recalcitrant tumor cell lines(1×10⁶ cells) with candidate viruses (MOI=1). Live cells willsubsequently be stained with JC1 vital dye to detect early apoptosisevents by dual color flow cytometry. Cells undergoing apoptosis will besorted onto monolayers of Vero cells to recover the virus replicatingwithin them. Iterative rounds of this assay, again with decreasingharvest times, will select for a more rapidly lethal phenotype. Virusesimproved in this way will be sequenced to map the genetic alterationsand contribute to our structure/function analysis efforts toward betterunderstanding of the biology of rhabdoviruses and oncolysis. The reversegenetic screen allows for an unbiased approach to improvingrhabdoviruses, and represents a good complement to efforts to makeimprovements through recombinant engineering of transgenes or rationalmutations based on structure/function studies.

Example 4 In Vivo Testing of Novel Recombinant Oncolytic Rhabdovirus(es)

The inventors have chosen to use orthotopic models of cancer as theymore accurately recapitulate the human clinical disease. However, unlikesubcutaneous tumor models, orthotopic tumors are not readily accessibleand therefore difficult to assess without sacrificing the experimentalanimal. To solve this problem, a multimodal optical imaging technologyis adopted that allows non-invasive imaging, and repeated measure thegrowth or regression of the implanted tumors, as well as the developmentor regression of distal metastatic lesions. The inventors have a highlysensitive fully integrated whole animal imaging platform (IVIS 200;Xenogen Corp) that can detect photons emitted even from within deeptissue. It can measure fluorescent light emitted by recombinantfluorescent proteins such as GFP as well as detect luciferase-generatedbioluminescence. By using substrate-specific luciferase reporter genes,one expressed from the virus and the other expressed from tumor cells,the inventors can measure the bioluminescence resulting from virusreplication concurrently with tumor measurements. To do this theinventors have cloned either YFP or a novel monomeric RFP in frame witheither firefly luciferase or a novel Renilla-like luciferase from themarine copepod Gaussia princeps. Between these two coding sequences theinventors have engineered a translation “stop-restart” sequence of 30amino acids. This small motif comes from the foot and mouth diseasevirus and allows for the stoichiometric expression of two proteins froma single mRNA, is very small and does not suffer from cell to cellvariability as do IRES motifs. These dual reporter constructs werecloned into lentivirus vectors, packaged into virus, and used toestablish stable reporter tagged 4T1, CT26 and U87 human glioblastomacells. These cells lines are used in three orthotopic mouse tumormodels: U87 human gliomas implanted intracranially into CD-1 nude mice;4T1 mouse breast carcinoma cells implanted into the fat pad of Balb/Cfemales (spontaneous, aggressive metastatic disease model); CT-26 coloncarcinoma injected into the tail vein of Balb/C mice (disseminatedtumors in the lung). The choice of orthotopic model was predicated onthe following criteria: aggressive, rapidly developing tumor, andtherefore challenging to treat; represent very different organ targets;span both immune competent and immunocompromised host systems.

The first studies will be to evaluate dose response characteristics inour models to identify an optimal dose. From preliminary toxicityexperiments, the inventors will have defined an MTD for each of ourcandidate strains in non-tumor bearing Balb/C animals. Therefore theinventors will test doses from the MTD, decreasing in half log intervalsdown to 1×10³ pfu. Using the IVIS to image replication in theestablished tumors, kinetics of initial virus delivery and duration ofsubsequent replication will be studied as a function of dose. Inparallel studies, mice will be sacrificed during this time course andexamined using fluorescence microscopy to determine how dose affects theability to reach all portions of the tumor and distal metastaticlesions. Healthy tissue will be examined to assess tumor specificreplication. Finally, safety at each dose will be determined bymonitoring mice for any signs of morbidity such as weight loss,dehydration, and behavioral changes. Tumor responses to the viruses inhead-to-head comparisons will be assessed following single dose IVtreatment. The sensitivity and quantitative nature of optical imagingtechnology make it ideally suited for this purpose. Thus tumors will beestablished as described above and monitor tumor growth or regressionfollowing virus dosing and compare these results to UV inactivated viruscontrols. Based on previous work with VSV, it is contemplated that asingle dose may not be sufficient for complete and durable tumorregressions. This necessitates a series of experiments to determine themost efficacious number and timing of doses. In a strategy similar tothat described above, the inventors will use tumor models to developmaximally effective dosing strategies. This will be done whilemonitoring for virus deliver to the tumor, replication, duration ofreplication at the tumor bed and spread to distant tumor sites, inconcert with tumor growth/regression. In addition, the inventors willexamine immune cell infiltration and activation in tumor beds andsurrounding lymph nodes using flow cytometry and immunohistochemistry asanother parameter of oncolytic activity. Ultimately, efficacy will beconfirmed by monitoring these mice for overall survival, and/or time toprogression; comparing virus treated groups with those treated withUV-inactivated virus as controls. An example of the animal model can befound in FIG. 13.

Cycle back to Optimization/Augmentation. It may be that several cyclesof optimization and then re-testing will be required to ultimatelydevelop a maximally effective therapeutic virus. Therefore, theinventors will use the results from in vivo testing to guide additionalrounds of biological and/or recombinant optimization and then re-test intumor models.

TABLE 4 Rhabdovirus mediated cell killing on the NCI 60 cell panel.Cells from the NCI 60 cell panel were plated in 6 well plates to aconfluency of 90%. These cells were infected at log dilutions withvarious rhabdoviruses, as indicated. After 48 hours, the monolayers werewashed, fixed and stained with crystal violet to score for viable cells.Values represent the pfu required to kill 50% of cells within 48 h.Malignancy Cell Line Chandipura Maraba Carajas Isfahan Klamath SawgrassVSV HR NSC LUNG A549-ATCC ≤10² ≤10² 10⁴ 10⁵ ≥10⁶ NE ≥10⁶ NSC LUNG EKVX≤10²   10³ ≥10⁶     10³ NSC LUNG HOP92   10³   10³ 10⁵ ≤10² NSC LUNGNCI-H226 ≥10⁶ ≥10⁶ 10⁴ NSC LUNG NCI-H23 ≤10² ≤10² ≤10²     10⁴ ≤10²MELANOMA LOX IMVI ≤10²   103 10³ ≤10² MELANOMA M 14   10³ ≤10² 10³ ≥10⁶  10⁵ MELANOMA SK-MEL-2 ≤10²   10³ ≤10² MELANOMA MALME 3M   10³   10⁵10⁵ 10³   10⁵ MELANOMA UACC-257 ≤10² ≤10² ≤10²   10³ ≤10² MELANOMAUACC-62 ≤10² 10³ ≥10⁶ LEUKEMIA MOLT-4   10³ ≤10² LEUKEMIA K-562   10⁵OVARIAN OVCAR-3   10³ ≤10² OVARIAN OVCAR-4   10³ ≤10² 10⁵ 10⁴ ≥10⁶   10⁴  10³ OVARIAN OVCAR-8 NE ≥10⁶ ≥10⁶   NE NE   10³ OVARIAN SK-OV-3 ≤10²  10⁵ 10⁵ ≥10⁶   ≥10⁶   10⁴ CNS SF-268 ≤10² 10⁴   10⁴ CNS SF-539 ≤10²≤10² 10³ 10⁴   10⁵ CNS SNB-19   10³   10⁴ ≤10²   ≤10² CNS SNB-75   10³  10³ NE 10⁵ ≥10⁶ ≤10² COLON HT29   10⁴ ≥10⁶ NE NE NE   10⁵ COLON COLO205 ≤10² ≤10² ≥10⁶     10³ COLON HCT-15   10⁵   10⁴ 10⁵ ≥10⁶     10³COLON SW-620 ≤10² ≤10² 10³ 10⁵ ≤10² BREAST HS 578T ≥10⁶ ≥10⁶ ≥10⁶   10⁴BREAST MDA-MB-435 ≤10² ≤10² ≤10²   10³ ≤10² RENAL TK-10 ≤10²   10³ 10⁴  10⁴ RENAL 786-O   10⁴ ≤10² 10⁵ 10⁵   10⁵ RENAL ACHN   10⁵   10³ 10⁵≥10⁶   NE ≤10² RENAL A498   10⁵   10⁵ ≥10⁶     10⁴ PROSTATE DU-145 ≤10²≥10⁶   ≥10⁶ PROSTATE PC-3 ≥10⁶ NE ≤10² MOUSE COLON CT26 ≤10² ≤10² ≥10⁶  NE ≤10²

TABLE 5 Focused comparison between four rhabdoviruses. Cells from theNCI 60 cell panel were plated in 6 well plates to a confluency of 90%.These cells were infected at log dilutions with various rhabdoviruses,as indicated. After 48 hours, the monolayers were washed, fixed andstained with crystal violet to score for viable cells. Values representthe pfu required to kill 50% of cells within 48 h. Chandipura MarabaCarajas WT VSV Lung A549 ≤10² ≤10²  10⁴ ≥10⁶ H226 ≥10⁶ ≥10⁶  10⁴ ≤10²melanoma M14  10³ ≤10²  10³  10⁵ Malme 3M  10³  10⁵  10⁵  10⁵ UACC-62≤10²  10³ ≥10⁶ leukemia K562  10⁵  10³ Ovarian OVCAR4  10³ ≤10²  10⁵ 10³ OVCAR8 ≥10⁶ ≥10⁶  10³ SK-OV-3 ≤10²  10⁵  10⁵  10⁴ CNS SF268 ≤10² 10⁴  10⁴ SF539 ≤10² ≤10²  10³  10⁵ Colon HCT-15  10⁵  10⁴  10⁵  10³Breast HS578T ≥10⁶ ≥10⁶  10⁴ Renal 786-O  10⁴ ≤10²  10⁵  10⁵ ACHN  10⁵ 10³  10⁵ ≤10² Prostate DU-145 ≤10² ≥10⁶ PC-3 ≥10⁶ ≤10²

Differences between VSV and other rhabdoviruses on the NCI 60 cell panelinclude: (1) preferential killing by Maraba virus compared to VSV ofA549 lung, M14 melanoma, UACC-62 melanoma, SF268 CNS, SF539 CNS, 786-Orenal, DU-145 prostate; (2) preferential killing by Carajas viruscompared to VSV for M14 melanoma, UACC-62 melanoma, SF539 CNS;preferential killing by VSV for H226 lung, K562 leukemia, OVCAR-8ovarian, HCT-15, HS578T breast, and PC-3 prostate. All other cell linesof the 60 cell panel show similar susceptibilities to VSV, Maraba andCarajas and Chandipura

TABLE 6 In vitro killing of selected transformed and immortalized cellsby novel rhabdoviruses. Cells were plated in 6 well dishes and allowedreach 75% confluency. These cells were subsequently infected with eachvirus at a fixed titer. Cultures were scored visually for cell deathafter 96 h. Muir Rio Le Farmington Springs Grande Ngaingan TibrogarganDantec Kwatta Human 293T ++++ ++++ +++ ++ + Mouse 4T1 + + ++ + HumanSW620 +++ +++ +++ + Hamster BHKT7 + +++ +++ +++ +++ Human U2OS ++++ ++++++ ++++ monkey Vero +++ ++++ +++ ++++ 4+ = 100% obliterated, 3+ =75-90% dead, 2+ = 50% dead, 1+ = <30% dead, −− = no death.

Example 5 Chimeric Rhabdoviruses

One potential problem with oncolytic viral compositions is the potentialfor an immune response in a patient. Such an immune response may bluntthe effectiveness of further applications of oncolytic virus since asignificant portion of the applied virus may be neutralized by thepatient's immune system. To avoid this problem is would be preferable tohave a plurality of oncolytic viral compositions that areimmunologically distinct. In this case a different oncolytic virus maybe applied to a patient for each subsequent therapy thereby providingsustained oncolytic activity that is minimally effected by a host immuneresponse. To this end a number of pseudotyped viral compositions wereconstructed and tested for their ability to infect cells.

To study the possibility of using oncolytic Rhabdoviruses that comprisesvarious G proteins from a number of Rhabdoviruses various recombinantviruses were constructed. Each recombinant included the VSV Indiana wildtype backbone (N, P, M and L genes) unless otherwise specified.Furthermore, recombinants included a luciferase reporter gene, eitherFirefly (FL) or Renilla (RL) between the G and the L gene. The generalnomenclature used to refer to the recombinants is RVR_(a)G^(x), whereinRVR stands for Rhabdovirus recombinant, (a) denotes the origin to theG-protein or G-protein-like gene and (x) denotes the version number.

RVR with Isfahan G protein. A RVR genome was cloned into the pXN2VSVvector such that XhoI and NheI restriction sites flanked the G or G-likegenes. The viral stop start sequence was added to the 3′ end of all G orG-like genes which encoded the following sequence:CTCGAGGGTATGAAAAAAACTAACAGATATCACGGCTAG (SEQ ID NO:25). Recombinantvirus was pseudotyped with the Isfahan G protein which has a proteinsequence identity of 37% compared to VSV G Ind. The RVR comprising theFL reporter gene was designated RVR_(Isf) (Isfahan) G¹ (wherein version1 indicates the presence of the FL reporter gene).

Furthermore antibody neutralization studies showed that serum comprisingantibodies from mice immunized with VSV WT did not significantlyneutralize the activity of RVR Isf G1 in vitro.

Furthermore, when mice immunized with VSV-WT were injected withRVR_(Isf)G¹ the virus with the Isf G polypeptide is able to evade theimmune system. As shown in FIG. 6C, RVR_(Isf)G¹ was detectable atvarious locations in immunized mice following viral inoculation. Thelevel of RVR_(Isf)G¹ detect in the immunized mice was similar to thelevel detected in naive controls animals (FIG. 6A). On the other hand,no virus was detected in immunized mice that were inoculated with VSV(FIG. 6B). Thus, oncolytic viruses comprising the Isf G polypeptideescape host immune response to previously administered VSV in vivo.

These results were further confirmed by injecting tumors in immunizednaïve mice with VSV or recombinant virus and determined the virus yieldfrom the infections. As shown in FIG. 7, recombinant virus injected intotumors of immunized or naïve mice yielded large amounts of progenyvirus. On the other hand, propagation of VSV injected in immunized micewas barely detectible.

Two additional RVRs comprising the Isf were also constructed.RVR_(Isf)G² comprises an RL reporter gene in place of the FL reportergene from RVR_(Isf)G¹. Also, RVR_(Isf)G³ comprises a chimeric VSV-Isf Gprotein. The chimeric protein (SEQ ID NO:19) comprises the Isfahan Gectodomain with VSV G transmembrane domain and cytoplasmic tail.

RVR with Chandipura G protein. Chandipura G has a protein sequencehomology of 42% with VSV G (Indiana). The same cloning strategydescribed above was used to construct RVR_(Cha)G¹. A one step growthcurve with RVR_(Cha)G¹ showed that it produces similar amounts of viruscompared to VSV (FIG. 8). Furthermore, the RVR had similar cytotoxicityas compared to VSV (FIG. 9).

RVR with Mamba G protein. Maraba G has a protein sequence homology 83%to VSV G (Indiana). Tins is the first report of the sequence of theMaraba G protein provided as a DNA sequence in SEQ ID NO:20. The samecloning strategy described above was used to construct RVR_(Mar)G¹. Aone step growth curve with RVR_(Mar)G¹ showed that recombinant virustiter was greater than VSV at 48 and 72 h. Thus, switching the G proteinmay stabilize the virus and thereby enhance yield (FIG. 10).Furthermore, the RVR_(Mar)G¹ was shown to be cytotoxic (FIG. 11).Furthermore, antibody neutralization assays showed that serum from miceimmunized with VSV WT did not neutralize the activity of RVR_(Mar)G¹indicating the RVR is capable of immune evasion.

RVR with Muir Springs G protein. Muir Springs G has 25.4% proteinsequence homology to VSV G (Indiana). The Muir Springs G sequence isprovided in SEQ ID NO:21 (amino acid) and SEQ ID NO:22 (DNA). The samecloning strategy described above was used to construct RVR_(Mur)G¹.

RVR with Klamath virus G protein. Pseudotyping experiments confirmedthat the Klamath G protein is functional at in a low pH (6.8)environment, unlike VSV G. This of great importance since it is knownthat the tumor core is hypoxic and acidic. Thus, it may be an advantageto have a virus which can replicate in such an environment. VSVHRGFP-Klamath pseudotyped were generated such that the virions containedthe genome of one virus but the envelope proteins of both viruses by coinfection into CT26 Cells. 24 hours after co infection the supernatantwas collected and the pseudotyped particles tittered. Pseudotyped viruswas then used (along with control virus to infect target cells in mediaof two different acidity. Results show that the Klamath G protein wasresponsible for the ability of the virus to infect at low pH.

Essentially the same cloning strategy described above was used toconstruct RVR_(Kla)G². However, unlike previous strategies, thisrecombinant includes the Klamath G in addition to the original VSV G(Indiana).

RVR with Farmington (Far) virus G protein. Farmington virus is anon-vesiculovirus that is non-neurotropic and demonstrates formation oflarge syncitia.

RVR with Bahia Grande (Bah) virus G protein. Bahia Grande virus is anon-vesiculovirus that is non-neurotropic.

RVR with JSR retroviral Env protein. Since VSV has a knownneurotoxicity, a strategy whereby a VSV recombinant would not infectneurons would be advantageous. JSR Env is originally from the JSRVretrovirus (a non-neurotropic virus) envelope (Env) genenon-neurotropic. A chimera comprising JSRV Env ectodomain with VSV Gtransmembrane domain and cytoplasmic tail is generated (DNA sequenceprovided as SEQ ID NO:23).

RVR with Ebola G protein. Ebola is a non-neurotropic virus with aglycoprotein that functions to bind receptor and mediate membranefusion. The G protein contains a furin Cleavage site at amino acidposition 497-501. The products of cleavage (GP1 & GP2) are linked bydisulfide bonds and thought to act as a possible decoy for neutralizingantibodies or immunomodulator. However, the furin cleavage site notrequired for infection or tropism. The Ebola G protein DNA sequence isprovided as SEQ ID NO:24.

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1. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an oncolytic recombinant rhabdovirus encoding a G protein from a first rhabdovirus and M, P, N and L proteins from a second rhabdovirus.
 2. The pharmaceutical composition of claim 1, wherein the G protein has at least about 85% amino acid sequence identity to an Isfahan virus G protein, a Chandipura virus G protein, a Maraba virus G protein, a Bahia Grande virus G protein, a Klamath virus G protein, or a Farmington virus G protein.
 3. The pharmaceutical composition of claim 2, wherein the oncolytic recombinant rhabdovirus encodes a G protein having at least about 85% amino acid sequence identity to an Isfahan virus G protein, a Chandipura virus G protein, a Maraba virus G protein, or a Muir Springs virus G protein.
 4. The pharmaceutical composition of claim 3, wherein the oncolytic recombinant rhabdovirus encodes a G protein having at least about 85% amino acid sequence identity to an Isfahan virus G protein, a Maraba virus G protein, or a Muir Springs virus G protein.
 5. The pharmaceutical composition of claim 4, wherein the oncolytic recombinant rhabdovirus encodes a G protein having at least about 85% amino acid sequence identity to a Maraba virus G protein.
 6. The pharmaceutical composition of claim 1, wherein the oncolytic recombinant rhabdovirus encodes M, P, N and L proteins from vesicular stomatitis virus (VSV).
 7. The pharmaceutical composition of claim 2, wherein the oncolytic recombinant rhabdovirus encodes M, P, N and L proteins from vesicular stomatitis virus (VSV).
 8. The pharmaceutical composition of claim 3, wherein the oncolytic recombinant rhabdovirus encodes M, P, N and L proteins from vesicular stomatitis virus (VSV).
 9. The pharmaceutical composition of claim 4, wherein the oncolytic recombinant rhabdovirus encodes M, P, N, and L proteins from vesicular stomatitis virus (VSV).
 10. The pharmaceutical composition of claim 5, wherein the oncolytic recombinant rhabdovirus encodes M, P, N, and L proteins from vesicular stomatitis virus (VSV).
 11. The pharmaceutical composition of claim 1, wherein said composition comprises 10³ to 10¹³ plaque forming units (pfu) of the oncolytic recombinant rhabdovirus.
 12. A method for treating cancer in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of claim
 1. 13. The method of claim 12, wherein the cancer is selected from the group consisting of lung cancer, head and neck cancer, breast cancer, cancer of the central nervous system, pancreatic cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, cervical cancer, ovarian cancer, gastrointestinal cancer, lymphoma, liver cancer, colon cancer, melanoma, and bladder cancer.
 14. The method of claim 12, wherein the cancer is metastatic.
 15. The method of claim 12, wherein the subject is a human.
 16. The method of claim 12, wherein the composition is administered by intraperitoneal, intravascular, intramuscular, intratumoral, subcutaneous or intranasal administration.
 17. The method of claim 16, wherein the composition is administered by intratumoral or intravascular administration.
 18. The method of claim 12, wherein the composition is administered multiple times.
 19. The method of claim 12, further comprising administering an additional anti-cancer therapy selected from the group consisting of chemotherapy, radiotherapy, and immunotherapy.
 20. A method for treating cancer in a subject comprising administering to the subject an effective amount of an oncolytic recombinant rhabdovirus encoding a G protein from a first rhabdovirus and M, P, N and L proteins from a second rhabdovirus.
 21. The method of claim 20, wherein the G protein has at least about 85% amino acid sequence identity to an Isfahan virus G protein, a Chandipura virus G protein, a Maraba virus G protein, a Bahia Grande virus G protein, a Klamath virus G protein, or a Farmington virus G protein.
 22. The method of claim 21, wherein the oncolytic recombinant rhabdovirus encodes M, P, N and L proteins from vesicular stomatitis virus (VSV).
 23. The method of claim 22, wherein the G protein has at least about 85% amino acid sequence identity to a Maraba virus G protein.
 24. The method of claim 20, wherein the cancer is selected from the group consisting of lung cancer, head and neck cancer, breast cancer, cancer of the central nervous system, pancreatic cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, cervical cancer, ovarian cancer, gastrointestinal cancer, lymphoma, liver cancer, lung cancer, colon cancer, melanoma, and bladder cancer. 